Methods for detecting human low molecular weight CD14

ABSTRACT

The present invention provides an antibody prepared using a peptide as an antigen, the peptide having 8 to 30 amino acid residues selected from an amino acid sequence at positions 1 to 68 of human high-molecular-weight CD14, or an antibody that binds to a peptide having a specific amino acid sequence at a position among the positions 1 to 68. An assay kit for human low-molecular-weight CD14 using the antibody and an assay method of the present invention, preferably a sandwich method, are able to quantitatively or qualitatively determine human low-molecular-weight CD14 with high sensitivity and specificity in a simple manner, so that they are useful for the diagnosis of a patient suffering from sepsis.

TECHNICAL FIELD

The present invention relates to an antibody prepared using a peptide asan antigen, the peptide having 8 to 30 amino acid residues selected fromspecific amino acid sequences for human high molecular weight CD14.Furthermore, the present invention relates to an antibody that binds toa peptide having a specific amino acid sequence in amino acid sequencesfor human high molecular weight CD14.

In addition, the present invention relates to an assay kit for humanlow-molecular-weight CD14 and a method of measuring the same.Furthermore, the present invention relates to a novel diagnostic methodfor sepsis in which human low-molecular-weight CD14 is determineddirectly. Furthermore, the present invention relates to a peptide usefulfor the preparation of the above antibody and a method of preparing theantibody.

BACKGROUND ART

A CD14 molecule was named as a protein identified by a family ofantibodies that recognize glycoproteins expressed on the membranesurface of monocytes in Third Leukocyte Typing Conference, 1986. In1990, Wright et al. elucidated that the CD14 molecule is a receptor forLPS, endotoxin (“Science”, vol. 249, p. 1431-1433, 1990). The CD14molecule is a glycoprotein having a molecular weight of 53-55 kDa, andanalyses on cDNA revealed that 1.4 kb mRNA has coding sequence of 356amino (“Nucleic Acids Research” (U.K.), vol. 16, p. 4173, 1988).

It was reported that human CD14 molecules include soluble CD14 moleculesin addition to membrane-bound CD14 molecules and blood contains solubleCD14 molecules having different molecular weights (“European Journal ofImmunology” (Germany), vol. 23, p. 2144-2151, 1993). In addition,Landmann et al. conducted Western blot analyses on soluble CD14 in serumof patients suffering from sepsis and reported that soluble CD14 ofabout 55 kDa is at high levels in non-survival sepsis patients andpatients with paroxysmal nocturnal hemoglobinuria (PNH) and that innormal sera, this molecule was not detected but soluble CD14 of 49-kDa,a slightly lower molecular weight than the former, was detected (“TheJournal of Infectious Disease”, vol. 171, p. 639-644, 1995).

Stelter reported that the difference in sugar chains is involved inthose subtypes having different molecular weights and two soluble CD14subtypes having different molecular weights are found in blood evenafter removal of N- and O-linked sugar chains (“European Journal ofBiochemistry” (Germany), vol. 236, p. 457-464, 1996). In addition,Bufler et al. conducted the C-terminal analysis on soluble CD14 andreported that a GPI group binds to a serine residue at position 327 ofsoluble CD14 and that a soluble CD14 molecule having a molecular weightof about 56 kDa is one of the molecular species from which GPI is notanchored (“European Journal of Immunology” (Germany), vol. 25, p.604-610, 1995).

Antibodies against CD14 molecules include many anti-CD14 antibodies,which have been prepared and used in identification of CD14 proteins,such as MEM-18 prepared by Bazil et al. (“European Journal ofImmunology” (Germany), vol. 16, p. 1583-1589, 1986), RoMo-1 prepared byShutt et al. (“Allergie und Immunologie” (Germany), vol. 34, p. 17-26,1988), and 3C10 prepared by Steinman et al. (“Journal of ExperimentalMedicine” (U.S.A.), vol. 158, p. 126-145, 1983).

Furthermore, soluble-CD14 assay systems using those antibodies have beenreported by Shutt et al. (DE-286876-A), Bazil et al. (“MolecularImmunology” (U.K.), vol. 26, p. 657-662, 1989), and Grunwald et al.(“Journal of Immunological Methods” (Holland), vol. 155, p. 225-232,1992), allowing the assay of soluble CD14 in human body fluid.

Furthermore, soluble CD14-ELISA kits have been released on the marketfrom IBL-Hanburg, Medgenix, and R & D Systems, and the assay of solubleCD14 has been performed for many diseases such as sepsis (“ClinicalImmunology And Immunopathology” (U.S.A.), vol. 80, p. 307-310, 1996; and“Rinshokensa”, vol. 38, p. 341-344, 1994),

However, it was found that soluble CD14 is not a sepsis-specific markerbecause of increases in levels of soluble CD14 molecules of about 55 kDaand 49 kDa (from report to report, the molecular weights are differentand not limited to about 55 kDa and 49 kDa, and the same will be appliedin the following description) depending on the degree of proceeding ofdiseases even in diseases except sepsis (“Infection and Immunity”(U.S.A.), vol. 67, p. 417-420, 1999; “Clinical and ExperimentalImmunology” (U.K.), vol. 120, p. 483-487, 2000; and “ClinicalExperimental Immunology” (U.K.), vol. 96, p. 15-19, 1994). Furthermore,the soluble CD14 was expected to be a marker for the severity of sepsis.However, the soluble CD14 has not been provided as a diagnostic productfor sepsis because of no correlation with septic shock (“Pediatricallergy and immunology) (Denmark), vol. 8, p. 194-199, 1997) and also nocorrelation with systemic inflammatory response syndrome (SIRS)(“European Journal of Clinical Investigation” (U.K.), vol. 28, p.672-678, 1998).

The inventors of the present invention have found out the presence of asoluble CD14 molecule with a low molecular weight of about 36 kDa inblood in addition to others such as two kinds of soluble CD14 moleculesdescribed above of about 55 kDa and 49 kDa reported by Landmann et al.(high molecular weight CD14 (from report to report, the molecularweights are different and not limited to about 55 kDa and 49 kDa, andthe same will be applied in the following description). The inventors ofthe present invention have also found out the presence of a small amountof the low-molecular-weight CD14 in normal individuals and of anincreased amount of the low-molecular-weight CD14 in patients sufferingfrom sepsis. Consequently, the inventors of the present invention havevalidated the clinical efficacy of the assay on a solublelow-molecular-weight CD14. However, the anti-CD14 antibodies known inthe art are those that recognize a high-molecular-weight soluble CD14protein or those recognize both high- and low-molecular-weight solubleCD14 proteins. Thus, no antibody that recognizes only alow-molecular-weight CD14 has been known in the art. Besides, the aminoacid sequence of the low-molecular-weight CD14 protein has beenconsidered to be identical with a part of the amino acid sequence of thehigh-molecular-weight soluble CD14 protein, so that the preparation ofan antibody as described above and a direct immunological assay on thelow-molecular-weight CD14 using the antibody have been considered to bedifficult. Therefore, as an assay for the soluble low-molecular-weightCD14, there is a proposal in which the level of low-molecular-weightCD14 in blood is indirectly obtained by subtracting the level ofhigh-molecular-weight CD14 in blood from the total level of the solubleCD14 in blood (International publication WO 01/22085).

DISCLOSURE OF THE INVENTION

Under such circumstances, an assay for qualitatively or quantitativelydetermining human low-molecular-weight CD14 with high sensitivity andspecificity in a convenient manner, the assay allowing directdetermination of the human low-molecular-weight CD14 and being usefulfor the diagnosis of a patient suffering from sepsis, and an assay kitfor the assay have been desired. Furthermore, an antibody against thehuman low-molecular-weight CD14 useful for the assay has been desired.

The inventors of the present invention have invented, as a result of theextensive study, an antibody prepared using a peptide as an antigen, thepeptide having 8 to 30 amino acid residues selected from amino acidsequences at positions 1 to 68 of human high-molecular-weight CD14 as anantibody which can be used for qualitatively or quantitativelydetermining human low-molecular-weight CD14 with high sensitivity andspecificity in a convenient manner. In addition, the inventors of thepresent invention have invented an antibody that binds to a peptidehaving a specific amino acid sequence in the amino acid sequence for thehuman high-molecular-weight CD14.

Furthermore, the inventors of the present invention have invented anassay for specifically determining human low-molecular-weight CD14 andan assay kit for human low-molecular-weight CD14. Still furthermore, theinventors of the present invention have invented a novel diagnosticmethod for sepsis in which human low-molecular-weight CD14 is determineddirectly. Besides, the inventors of the present invention have inventeda peptide useful for the preparation of the above antibody and a methodof preparing the above antibody.

In other words, the present invention provides the following novelantibodies and an assay kit for human low-molecular-weight CD14.

-   (1) Antibodies as described in the following (1-1) to (1-5):-   (1-1) An antibody prepared using a peptide as an antigen, the    peptide having consecutive 8 to 30 amino acid residues selected from    an amino acid sequence described in SEQ ID NO: 1.-   (1-2) An antibody prepared using a peptide as an antigen, the    peptide having consecutive 8 to 20 amino acid residues selected from    an amino acid sequence described in SEQ ID NO: 1.-   (1-3) An antibody prepared using a peptide as an antigen, the    peptide having consecutive 8 to 16 amino acid residues selected from    amino acid sequences at positions 53 to 68 in the amino acid    sequence described in SEQ ID: 1.-   (1-4) An antibody prepared using a peptide as an antigen, the    peptide having amino acid residues described in any one of SEQ ID    NOS: 2 to 4.-   (1-5) An antibody prepared using a peptide as an antigen, the    peptide having 16 amino acid residues described in SEQ ID NO: 2.-   (2) Antibodies as described in the following (2-1) and (2-2):-   (2-1) An antibody that binds to a peptide having amino acid residues    described in any one of SEQ ID NOS: 2 to 4.-   (2-2) An antibody that binds to a peptide having 16 amino acid    residues described in SEQ ID NO: 2.-   (3) Assay kits for human low-molecular-weight CD14, represented in    the following (3-1) to (3-22):-   (3-1) An assay kit for human low-molecular-weight CD14 for directly    assaying human low-molecular-weight CD14 in a specimen without    detecting human high-molecular-weight CD14, comprising an antibody    that binds to at least one of the human low-molecular-weight CD14 or    a fragment thereof.-   (3-2) The assay kit for human low-molecular-weight CD14 of (3-1),    wherein the antibody that binds to the human low-molecular-weight    CD14 or the fragment thereof is the antibody described in any one of    the above (1-1) to (1-5), (2-1), or (2-2), or a fragment thereof.-   (3-3) The assay kit for human low-molecular-weight CD14 of (3-1),    wherein the antibody that binds to the human low-molecular-weight    CD14 or the fragment thereof is the antibody described in any one of    the above (1-4), (1-5), (2-1), or (2-2), or a fragment thereof.-   (3-4) The assay kit for human low-molecular-weight CD14 of (3-1),    wherein the antibody that binds to the human low-molecular-weight    CD14 or the fragment thereof is the antibody described in any one of    the above (1-5) or (2-2), or a fragment thereof.-   (3-5) The assay kit for human low-molecular-weight CD14 of any one    of (3-1) to (3-4), wherein the human low-molecular-weight CD14 is    assayed by a sandwich immunoassay method.-   (3-6) The assay kit for human low-molecular-weight CD14 of (3-5),    further comprising a second binding substance that binds to the    human low-molecular-weight CD14.-   (3-7) The assay kit for human low-molecular-weight CD14 of (3-6),    wherein the second binding substance is an antibody that binds to    the human low-molecular-weight CD14 or a fragment thereof.-   (3-8) The assay kit for human low-molecular-weight CD14 of (3-6),    wherein the second binding substance is a monoclonal antibody that    binds to the human low-molecular-weight CD14.-   (3-9) The assay kit of (3-6), wherein the second binding substance    is an antibody that binds to any one region of: amino acid residues    at positions 1 to 52 of human high-molecular-weight CD14; a fragment    thereof; an antibody that competes with or shows cross-reactivity    with an antibody that binds to any one region of amino acid residues    at positions 1 to 52 of the human high-molecular-weight CD14: or a    fragment thereof.-   (3-10) The assay kit of (3-6), wherein the second biding substance    is: an antibody that binds to any one of amino acid residues at    positions 17 to 26 of human high-molecular-weight CD14; a fragment    thereof; an antibody that competes with or shows cross-reactivity    with an antibody that binds to any one of amino acid residues at    positions 17 to 26 of the human high-molecular-weight CD14; or the    fragment thereof.-   (3-11) The assay kit for human low-molecular-weight CD14 of any one    of (3-6) to (3-10), wherein the antibody described in any one of the    above (1-1) to (1-5), (2-1), or (2-2), or a fragment thereof is    binding to an insoluble carrier.-   (3-12) The assay kit for human low-molecular-weight CD14 of any one    of (3-6) to (3-10), wherein the second binding substance is binding    to an insoluble carrier.-   (3-13) The assay kit for human low-molecular-weight CD14 of any one    of (3-6) to (3-10), wherein the antibody described in any one of the    above (1-1) to (1-5), (2-1), or (2-2), or a fragment thereof is    labeled.-   (3-14) The assay kit for human low-molecular-weight CD14 of any one    of (3-6) to (3-11), wherein the second binding substance is labeled.-   (3-15) The assay kit for human low-molecular-weight CD14 of any one    of (3-6) to (3-14), further comprising: a second specific binding    substance and a partner of the second specific binding substance,    wherein the second specific binding substance and the partner    thereof form second specific binding together.-   (3-16) The assay kit for human low-molecular-weight CD14 of (3-15),    wherein the second specific binding substance or the partner thereof    is binding to an insoluble carrier.-   (3-17) The assay kit for human low-molecular-weight CD14 of (3-15),    wherein the second specific binding substance or the partner thereof    is labeled.-   (3-18) The assay kit for human low-molecular-weight CD14 of any one    of (3-5) to (3-12), (3-15), and (3-16), further comprising a labeled    human low-molecular-weight CD14 or a labeled human    low-molecular-weight CD14 analogue, wherein the assay is performed    by a sandwich immunoassay based on a competition method.-   (3-19) The assay kit for human low-molecular-weight CD14 of any one    of (3-5) to (3-18), wherein the label is at least one of an enzyme,    a dyestuff, a gold colloid, a colored latex, a chemiluminescent    substance, a fluorescent substance, and an isotope.-   (3-20) The assay kit for human low-molecular-weight CD14 of any one    of (3-5) to (3-19), wherein the sandwich immunoassay method is an    assay method utilizing immunochromatography.-   (3-21) The assay kit for human low-molecular-weight CD14 of any one    of (3-5) to (3-19), wherein the sandwich immunoassay method is an    assay method utilizing a flow-through method.-   (3-22) The assay kit for human low-molecular-weight CD14 of (3-1),    wherein the assay is performed by an agglutination method, a direct    solid-phase method, or a competition method.

Furthermore, the following assay for human low-molecular-weight CD14,novel diagnostic method for sepsis, peptide, and method of preparing anantibody are provided:

-   (4) Assays for human low-molecular-weight CD14 as described in the    following (4-1) to (4-3).-   (4-1) An assay method for human low-molecular-weight CD14, which is    for directly assaying human low-molecular-weight CD14 in a specimen    using an antibody that binds to at least one of the human    low-molecular-weight CD14 in order to detect the human    low-molecular-weight CD14 without detecting human    high-molecular-weight CD14.-   (4-2) The assay method for human low-molecular-weight CD14 of (4-1),    wherein the antibody that binds to the human low-molecular-weight    CD14 is the antibody described in any one of the above (1-1) to    (1-5), (2-1), or (2-2), or a fragment thereof.-   (4-3) The assay for human low-molecular-weight CD14 of (4-2),    wherein the human low-molecular-weight CD14 is determined by a    sandwich immunoassay.-   (5) A diagnostic method for sepsis, which is for directly assaying    human low-molecular-weight C14.-   (6) A peptide having amino acid residues described in any one of SEQ    ID NOS: 2 to 4.-   (7) The methods of preparing antibodies as described in the    following (7-1) and (7-2).-   (7-1) A method of preparing the antibody of any one of the above    (1-1) to (1-5), (2-1), and (2-2), wherein a peptide having    consecutive 8 to 30 amino acid residues selected from the amino acid    sequence described in SEQ ID NO: 1, or a peptide having amino acid    residues described in any one of SEQ ID NOS: 2 to 4 is used as an    antigen.-   (7-2) A method of preparing an antibody of any one of (1-4), (1-5),    (2-1), and (2-2) wherein a peptide having amino acid residues    described in any one of SEQ ID NOS: 2 to 4 is used an antigen.

The antibody of the present invention can be used in the assay kit forhuman low-molecular-weight CD14 of the present invention, and the kitallows qualitative or quantitative measurement of humanlow-molecular-weight CD14 with high sensitivity and specificity in aconvenient manner and is useful for diagnosis of a patient sufferingfrom sepsis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of an immunochromatography kit using anS68-peptide polyclonal antibody, in which part (A) is a schematicdiagram of immunochromatographic assay using a gold-colloid-labeledF1031-8-3 as a labeled antibody and part (B) is a schematic diagram ofan immunochromatography kit using biotin and streptavidin as secondbinding substances.

FIG. 2 shows the results of the assay performed on a standard substanceby an immunochromatography kit using the S68-peptide polyclonalantibody.

FIG. 3 shows the results in which only an S68 peptide inhibits thebinding between the S68-peptide polyclonal antibody and alow-molecular-weight CD14 protein, in which part (A) shows a state whereno binding is found in the sera of normal individuals and part (B) showsthe inhibition of binding with the S68 peptide in the sera of patientssuffering from sepsis.

FIG. 4 is a diagram that represents a standard curve obtained by an EIAkit for low-molecular-weight CD14 of the present invention using ansCD14 (1-307) S286C protein.

FIG. 5 is a diagram illustrating a case where a soluble CD14 proteinderived from the serum of a normal individual does not affect the valuesmeasured by the EIA kit for low-molecular-weight CD14 of the presentinvention using the sCD14 (1-307) S286C.

FIG. 6 is a diagram showing the results obtained by analyzing thelow-molecular-weight CD14 protein and the high-molecular-weight CD14protein in the sera of patients suffering from sepsis by using the EIAkit for low-molecular-weight CD14 and the commercially-availableCD14-EIA kit (IBL-Hamburg), respectively, with gel filtrationchromatography.

FIG. 7 is a diagram showing the results obtained by analyzing thelow-molecular-weight CD14 protein and the high-molecular-weight CD14protein in the sera of patients suffering from sepsis by using the EIAkit for low-molecular-weight CD14 and the commercially-availableCD14-EIA kit (IBL-Hamburg), respectively, with gel filtrationchromatography, in which black arrows on the upper side of the figurerespectively indicate the positions of markers used for calibration,i.e., from the left side, BSA, ovalbumin, chymotrypsinogen-A, andribonuclease-A.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in more detail.

Major soluble CD14 molecules in human blood include soluble CD14molecules of about 55 kDa and about 49 kDa (hereinafter, “human” may beomitted and they may be described as high-molecular-weight CD14molecules) described in the report by Landmann et al. described inBackground Art. It is confirmed that those high-molecular-weight CD14molecules bind to an F1025-3-1 antibody (see WO 01/22085).

On the other hand, it is also found that there is a CD14 fragment whichdoes not bind to the F1025-3-1 antibody, in other words, thelow-molecular-weight CD14 which has lower molecular-weight is presentother than the high-molecular-weight CD14. An increase in level of thelow-molecular-weight CD14 in blood with respect to a specific disease isalso shown (see WO 01/22085).

Hereinafter, a description will be given of the humanlow-molecular-weight CD14 (hereinafter, “human” may be omitted and itmay be described as low-molecular-weight CD14) provided as an analyte ofthe assay in the present invention.

The human low-molecular-weight CD14 provided as an analyte of the assayin the present invention has at least three characteristic features asfollows:

-   (1) no binding to an F1025-3-1 antibody;-   (2) specific binding to an antibody prepared using a peptide as an    antigen, the peptide having 16 amino acid residues described in SEQ    ID NO: 2; and-   (3) showing a peak in the molecular weight range of 25 to 45 kDa on    gel filtration chromatography.

The characteristic feature (1) described above allows the humanlow-molecular-weight CD14 provided as an analyte of the assay in thepresent invention to be recognized as a molecule different from thehigh-molecular-weight CD14 described above. The F1025-3-1 antibodydescribed in the characteristic feature (1) is an antibody preparedusing a peptide as an antigen, the peptide having the amino acidsequences at positions 316 to 328 of the full-length human CD14described in SEQ ID NO: 5. Thus, because of no binding to the F1025-3-1antibody, it is conceivable that the sequences at positions 316 andlater of the full-length human CD14 described in SEQ ID NO: 5 are notexistent in the human low-molecular-weight CD14.

The peptide having 16 amino acids described in SEQ ID NO: 2 described inthe above characteristic feature (2) corresponds to 16 amino acidresidues at positions 53 to 68 of the human CD14 described in SEQ ID NO:5. Among human proteins, except human CD14, other proteins that containthe sequence of SEQ ID NO: 2 have not been known up to now, so that thesequence may be a sequence specific to the human CD14. This factconfirms that the peptide provided as an analyte of the assay in thepresent invention can be one kind of human CD14.

As the characteristic feature (2′) instead of the characteristic feature(2), furthermore, human low-molecular-weight CD14 as an analyte of theassay in the present invention may be characterized by biding to anantibody that binds to a peptide having 16 amino acid residues describedin SEQ ID: 2.

From the above characteristic feature (3), the humanlow-molecular-weight CD14 as an analyte of the assay in the presentinvention shows the peak of elution in the molecular weight range of 25to 45 kDa by gel filtration chromatography. In general, amolecular-weight analysis with gel filtration chromatography causevariations in the results of the assay depending on the experimentalconditions including a resin used for chromatography, the dimensions ofa column, and the molecular weight of a marker used. The humanlow-molecular-weight CD14 as an analyte of the assay in the presentinvention is characterized in that it can be distinguished from humanhigh-molecular-weight CD14 in gel filtration chromatography and can beeluted at lower molecular weights.

The human low-molecular-weight CD14 having characteristic features,which can be explained by the above (1) to (3), is provided as ananalyte of the assay in the present invention. Further preferablecharacteristic features of the human low-molecular-weight CD14 as ananalyte of the assay in the present invention will be described below.

(4) Specific binding to an anti-human CD14 polyclonal antibody.

The human low-molecular-weight CD14 as an analyte of the assay in thepresent invention specifically binds to a polyclonal antibody using thefull-length human CD14 or recombinant full-length human CD14 as anantigen. Examples of anti-human CD14 polyclonal antibodies include:antisera having increased antibody titers obtained by immunizing micewith CD14 proteins in human blood as described later in Example3-(2)-[2]; and specific antibodies included therein.

Furthermore, the human low-molecular-weight CD14 is characterized bybinding to a specific anti-CD14 monoclonal antibody. For example, thehuman low-molecular-weight CD14 is particularly characterized in that itbinds to an anti-CD14 monoclonal antibody recognizing an amino acidsequence at positions 17 to 26 of the full-length human CD14 describedin SEQ ID NO: 5 or to an anti-CD14 monoclonal antibody that competeswith the antibody. Concrete examples of such an antibody include anF1031-8-3 antibody described below prepared using CD14 in human serum asan antigen and an F1106-13-3 antibody described below prepared usingrecombinant human CD14 as an antigen.

On the other hand, the human low-molecular-weight CD14 is furthercharacterized as follows. The human low-molecular-weight CD14 ischaracterized in that it may bind to: one of an anti-CD14 monoclonalantibody that recognizes an amino acid sequence at positions 17 to 26 ofthe full-length human CD14 described in SEQ ID NO: 5 and an anti-CD14monoclonal antibody that competes with the antibody; and an antibodyprepared using a peptide as an antigen, the peptide having 16 amino acidresidues described in SEQ ID NO: 2, as described in the abovecharacteristic feature (2) concurrently at two positions. For example,the human low-molecular-weight CD14 is characterized in that it may beassayed by a sandwich method using a combination of these twoantibodies.

The inventors of the present invention have found that the humanlow-molecular-weight CD14 explained in the above description is asoluble protein in human blood and exists more in blood of patientssuffering from sepsis compared with normal individuals and that theprotein can be directly assayed using a specific antibody. By the way,in WO 01/22085 described above, a protein having a molecular weight of36 kDa is exemplified as one of molecular species of thelow-molecular-weight CD14 as an analyte of the assay.

By the way, the “soluble CD14” described in the present specificationmeans a protein existed in human plasma and is used in contrast with the“membrane-bound CD14” which is attached on a cell membrane but not foundin human plasma.

The “antibody prepared using a peptide as an antigen” used in thepresent invention means an antibody in which a peptide used as an“antigen” is provided as an epitope or a part of an epitope. Inaddition, it is an antibody that shows an ability of binding to apeptide used as an “antigen” of the “antibody prepared using a peptideas an antigen”. The examples of the “antibody prepared using a peptideas an antigen” include antibodies that represent the propertiesdescribed above even though the antibodies are prepared using peptidesas their immunogens with addition of carriers or carrier proteins orother amino acid residues for providing the respective peptides as“antigens” with immunogenicity.

According to a first aspect of the present invention, there is providedan antibody prepared using a peptide as an antigen, the peptide havingconsecutive 8 to 30 amino acid residues selected from the amino acidsequence described in SEQ ID: 1.

An antibody according to the first aspect of the present invention isprepared using a peptide as an antigen, the peptide having consecutive 8to 30 amino acid residues selected from the amino acid sequencedescribed in SEQ ID: 1.

The number of amino acid residues is not specifically limited as far asit is an antibody prepared using a peptide as an antigen, the peptidehaving consecutive 8 to 30 amino acid residues selected from the aminoacid sequence described in SEQ ID NO: 1. It is an antibody preparedusing a peptide as an antigen, the peptide having preferably 10 or moreconsecutive amino acids, more preferably 12 or more consecutive aminoacids, particularly preferably 16 consecutive amino acids. In addition,it is an antibody prepared using a peptide as an antigen, the peptidehaving preferably 25 or less, more preferably 20 or less amino acids.

Furthermore, it may be any one region at positions 1 to 68 in the aminoacid sequence described in SEQ ID NO: 1 and is not particularly limited.However, preferable is an antibody prepared using a peptide as anantigen, the peptide having consecutive 8 to 16 amino acid residuesselected from the amino acid sequences at positions 53 to 68 in theamino acid sequence described in SEQ ID NO: 1. In addition, antibodiesprepared using peptides as antigens, the peptide having amino acidresidues described in SEQ ID NO: 2-4, respectively, are preferable. SEQID NO: 5 describes the amino acid sequence of a full-length human CD14.The amino acid sequence described in SEQ ID NO: 1 corresponds topositions 1 to 68 of the amino acid sequence described in SEQ ID: 5.Furthermore, the amino acid sequences described in SEQ ID NOS: 2, 3, and4 correspond to positions 53 to 68 (16 amino acid residues), 1 to 17 (17amino acid residues), and 14 to 32 (19 amino acid residues) of the aminoacid sequence described in SEQ ID NO: 5, respectively. That is, each ofthe amino acid residues described in SEQ ID NOS: 2 to 4 is consecutiveamino acid residues included in the amino acid sequence described in SEQID NO: 1.

More preferably, it is an antibody prepared using a peptide as anantigen, the peptide having 16 amino acid residues described in SEQ IDNO: 2.

The characteristic feature of the antibody according to the first aspectof the present invention is to bind to human low-molecular-weight CD14.This feature allows the antibody to be used in a kit according to afourth aspect of the present invention or an assay according to a fifthaspect of the present invention.

Furthermore, the molecular weight of the human low-molecular-weight CD14is different from that of the high-molecular-weight CD14, and also theamino acid sequence of the former is shorter than that of the latter.For this reason, the conformation of the low-molecular-weight CD14 inblood is different from that of the high-molecular-weight CD14, so thattheir reactivity with the antibody may be different from each other.Thus, it is conceivable that the antibody according to the first aspectof the present invention strongly binds to the low-molecular-weightCD14.

According to a second aspect of the present invention, there is providedan antibody that binds to a peptide having amino acid residues describedin any one of SEQ ID NOS: 2 to 4.

The antibody according to the second aspect of the present invention maybind to any region of a peptide, which is not specifically limited asfar as it binds to a peptide having amino acid residues described in anyone of SEQ ID NOS: 2 to 4.

Preferably, it is an antibody that binds to a peptide having 16 aminoacid residues described in SEQ ID NO: 2.

The characteristic feature of the antibody according to the secondaspect of the present invention is to bind to the humanlow-molecular-weight CD14. This feature allows the antibody to be usedin a kit according to the fourth aspect of the present invention or anassay according to the fifth aspect of the present invention.

Furthermore, the molecular weight of the human low-molecular-weight CD14is different from that of the high-molecular-weight CD14, and also theamino acid sequence of the former is shorter than that of the latter.For this reason, the conformation of the low-molecular-weight CD14 inblood is different from that of the high-molecular-weight CD14, so thattheir reactivity with the antibody may be different from each other.Thus, it is conceivable that the antibody according to the second aspectof the present invention strongly binds to the low-molecular-weightCD14.

The phrase “binding to a peptide having amino acid residues described inany one of SEQ ID NOS: 2 to 4” means that the antibody specificallybinds to a peptide as an antigen, the peptide having amino acid residuesdescribed in each of SEQ ID Numbers and shows a normal antigen-antibodyreaction. For instance, the phrase “binding to a peptide having 16 aminoacid residues described in SEQ ID NO: 2” means that the antibodyspecifically binds to a peptide as an antigen, the peptide having 16amino acid residues described in SEQ ID NO: 2 and shows a normalantigen-antibody reaction. The representation of the antibody-antigenreaction can be identified by a agglutination method, sandwich method,solid-phase direct method or solid-phase binding method, competitionmethod, and so on.

The dissociation constant (KD) of the antibody according to the secondaspect of the present invention is, when the dissociation constant isexpressed as affinity to the peptide or the low-molecular-weight CD14,preferably less than 10⁻⁷ M, more preferably 10⁻⁸ M or less, still morepreferably 10⁻⁹ M or less.

In the preparation of the antibody according to the second aspect of thepresent invention, the peptide used as the antigen is a peptide thatcontains consecutive 8 or more amino acids of the amino acid residuedescribed in any one of SEQ ID NOS: 2 to 4, preferably consecutive 10 ormore, more preferably consecutive 12 or more, particularly preferablyconsecutive 16 or more amino acids. Furthermore, as far as the peptidecontains consecutive 8 or more amino acids of the amino acid residuedescribed in any one of SEQ ID NOS: 2 to 4, the other amino acidsequences are not limited. The entire amino acid sequence of the peptideis preferably derived from an amino acid sequence described in any oneof SEQ ID NOS: 2 to 4.

The antibody according to the second aspect of the present invention ispreferably an antibody prepared using a peptide as an antigen, thepeptide having consecutive 8 or more amino acids of amino acid residuesdescribed in SEQ ID NO: 2. It is an antibody prepared using a peptide asan antigen, the peptide having preferably consecutive 10 or more, morepreferably consecutive 12 or more, particularly preferably consecutive16 or more amino acids.

The antibody according to the first aspect of the present invention andthe antibody according to the second aspect of the second aspect of thepresent invention (hereinafter, they may be described as the antibodiesof the present invention) may be polyclonal antibodies or monoclonalantibodies. The species of an animal from which the antibody of thepresent invention is originated are not specifically limited. In termsof facilitating the preparation of the antibody, a rabbit, goat, or thelike is preferable. In addition, the immunoglobulin species may be usedin any one of classes, subclasses, and isotypes.

Examples of a method of preparing a peptide to be used as an immunogeninclude a method using a generally-employed peptide synthesizer (PeptideSynthesizer 433A Type, Perkin-Elmer, Japan) or the like and a geneticrecombination method (“New Cell Engineering Experiments Protocols,” Ed.Department of Carcinostatic Research, The Institute of Medical Science,The University of Tokyo, Shujunsha).

For instance, a peptide having consecutive 8 or more amino acids ofamino acid residues described in SEQ ID NO: 2 can be synthesized by anFmoc method using a 433A Type peptide synthesizer. After deprotectionwith TFA and cutting out from the resin, the resultant is purified byusing a C18 HPLC column (Capcell-pak, Shiseido Co., Ltd.), to therebyprepare the target peptide.

When the antigen is a protein, it can be directly used as an immunogen.However, when a peptide has 8 to 30 amino acid residues or less, themolecular weight of the peptide is small, it is not enough to use, asimmunogen, in general. In this case, the peptide may be provided as anantigen by binding the peptide to a carrier or by using a MultipleAntigen Peptide (MAP) method. Then, an MAP peptide is prepared andprovided with a molecular weight that allows the antigen to haveimmunogenicity.

Carriers to be bound to the peptides described above include carrierproteins and polymers. The carrier proteins used may be heteroproteinssuch as bovine serum albumin, keyhole limpet hemocyanin (KLH),thyroglobulin, and ovalbumin. Those carrier proteins utilize thefunctional groups of the side chain in an amino acid of a peptide orcarrier protein or introduce a maleimide group, N-hydroxysuccinimide(NHS) group, or aldehyde group to allow the carrier to bind to the abovepeptide. Examples of the polymers include saccharides such as mannan andchitosan and polyvinylpyrrolidone (PVA). Those polymers may bind to theabove peptides by means of adsorption or chemical binding as describedabove.

The antibody of the present invention can be prepared using thetechnologies known in the art (see, for example, Procedures ofImmunological Experiments, The Japanese Society for Immunology, Ed.,published by The Japanese Society for Immunology). For instance, apolyclonal antibody can be prepared by the following method.

Any one of the various animals can be immunized by a mixture of 20 to1000 μg of immunogen prepared as described above with an adjuvant suchas a Freund's complete adjuvant, RIBI adjuvant, or ALUM. Examples of thevarious animals which can be used include a horse, sheep, goat, pig,rabbit, rat, and mouse. Immunization procedures which can be usedinclude intramuscular administration, intradermal administration,subcutaneous administration, intraperitoneal administration, andlymph-node administration. A booster immunity can be given such that,every 1-4 weeks after first time administration, the immunogen mixedwith the adjuvant such as a Freund's incomplete adjuvant, RIBI adjuvant,or ALUM is administrated similarly or the immunogen is intravenouslyadministrated in a direct manner. An antiserum can be prepared from animmunized animal by a normal blood sampling method, for example, amethod including: collecting blood from the carotid artery, auris vein,heart, leg vein, or the like; and separating the serum from the blood bymeans of centrifugation or the like. The resulting antiserum issubjected to a salting-out method involving the addition of ammoniumsulfate, sodium sulfate, or the like to precipitate a γ-globulinfraction. Then, after the fraction has been dialyzed in an appropriatebuffer, a purified polyclonal antibody of the fraction of IgG against atarget peptide can be prepared using an affinity matrix of protein A,protein G, or the like capable of specifically purifying γ-globulin. Inaddition, specific purification can be performed by selecting anantibody that binds to the above antigen.

Furthermore, the monoclonal antibody can be prepared by the followingmethod.

The antibody of the present invention can be prepared by: fusingimmunocytes of an immunized animal with myeloma cells to preparehybridomas; and selecting a clone that produces an antibody capable ofbinding to the above peptide from the hybridomas. Preferably, animmunogen is a peptide having consecutive 10 or more amino acid residuesat positions 53 to 68. In addition, an immunogen is more preferably apeptide having consecutive 12 or more amino acids, particularlypreferably a peptide having consecutive 16 amino acids.

Although a mammal to be immunized is not specifically limited, it ispreferably selected in consideration of compatibility with myeloma cellsto be used in cell fusion and preferably a mouse, rat, hamster, or thelike. The myeloma cells which can be used are various kinds of cellswell known in the art including myeloma cells P3, P3U1, SP2/O, NS-1,YB2/0, and Y3-Ag 1, 2, and 3.

The immunization can be performed by a known method. For example, theimmunization is performed by administering an antigen intraperitoneally,subcutaneously, intravenously, or into the foot pad. The antigen may beadministered in combination with an adjuvant and it is preferable toadminister the antigen in a plurality of times. The immunocytes arepreferably spleen cells or cells derived from lymph node isolatedseveral days, for example, 3 days after the final administration of theantigen. Immunocytes and myeloma cells can be fused using a known methodsuch as the method of Milstein et al. (Methods in Enzymol., vol. 73, p.3). For example, mention may be made of the method using polyethyleneglycol (PEG) as a fusing agent, an electric field-induced cell fusionmethod, or the like. A mixing ratio of immunocytes and myeloma cells isnot particularly limited as far as it allows the fusion. However, it ispreferable to make the amount of myeloma cells 1/10 to the equivalentrelative to immunocytes. In the method in which cell fusion is performedusing PEG (mean molecular weight: 1,000 to 4,000), the concentration ofPEG is not particularly limited. However, it is preferable that fusionbe performed at a concentration of 50%. An auxiliary such as dimethylsulfoxide (DMSO) may be added as an enhancer of the fusion efficiency.The fusion is started by the addition of a PEG solution warmed at 37° C.to mixed cells and is terminated by the addition of a culture mediumafter reacting the solution and the cells for 1 to 5 minutes. Thehybridomas created by the fusion are incubated for 1 to 7 days in aselection medium such as a culture medium containing hypoxanthine,thymidine, and aminopterin (HAT medium) to separate them from nonfusedcells.

The obtained hybridomas are further selected by antibodies produced bythem. A selected hybridoma is converted into a monoclonal by a knownlimiting dilution method to establish a monoclonal antibody producinghybridoma. Any one of the known methods may be used as the method ofdetecting the activity of an antibody that the hybridoma produces.Examples of the methods include an ELISA, agglutination reaction, andradio immunoassay. Examples of the established hybridoma may becultivated by a known method and a monoclonal antibody may be obtainedfrom the culture supernatant. In addition, the hybridoma is administeredto a mammal having compatibility therewith to allow proliferation, andthe proliferated hybridoma is obtained from the ascites. Purification ofthe antibody can be performed using a known purification method such assalting out, gel filtration, ion exchange chromatography, or affinitychromatography.

Furthermore, as described in the aspect below, the antibody of thepresent invention can be used in the assay kit for humanlow-molecular-weight CD14 of the present invention, it is conceivablethat an antibody that binds to the human low-molecular-weight CD14 butnot to human high-molecular-weight CD14 may be prepared.

It is conceivable that the antibody that binds to the humanlow-molecular-weight CD14 but not to human high-molecular-weight CD14may be obtained by preparing an antibody using the low-molecular-weightCD14 as an antigen and selecting an antibody that does not bind to thehigh-molecular-weight CD14.

A method of preparing the low-molecular-weight CD14 is described inExample 16 of WO 01/72993. In addition, using the antibody of thepresent invention, the low-molecular-weight CD14 can be prepared byspecific purification from human serum, preferably from the serum of apatient suffering from sepsis.

For selecting the antibody that does not bind to thehigh-molecular-weight CD14, the binding between the resulting antibodyand the high-molecular-weight CD14 may be assayed by a agglutinationmethod, sandwich method, solid-phase direct method or solid-phasebinding method, competition method, or the like. Those methods will bedescribed later.

The high-molecular-weight CD14 may be prepared using an antibodyspecific to the high-molecular-weight CD14, which is described inExample 5 of WO 01/22085.

It is also conceivable that the antibody may be prepared by preparingand selecting an antibody that does not bind to thehigh-molecular-weight CD14 by the same way as described above using apeptide as an antigen, the peptide having a part of the amino acidsequence of human CD14. The “peptide having a part of the amino acidsequence of human CD14” means, for example, each peptide that containsconsecutive 8 or more amino acids in the sequence of 16 amino acidsdescribed in SEQ ID NO: 2.

According to a third aspect of the present invention, there is provideda assay kit for human low-molecular-weight CD14, which contains anantibody that binds to at least one of human low-molecular-weight CD14or a fragment of the antibody and which directly assays the humanlow-molecular-weight CD14 in a specimen without detecting humanhigh-molecular-weight CD14.

The kit of the present invention contains an antibody that binds to atleast one of human low-molecular-weight CD14 or a fragment of theantibody and directly assays the human low-molecular-weight CD14 in aspecimen. In addition, the kit detects the human low-molecular-weightCD14 as an analyte but does not detect human high-molecular-weight CD14,so that the human low-molecular-weight CD14 can be directly assayed. The“fragment of the antibody” means Fab, Fab′, or F(ab′)₂ of the antibody.

The assay kit for human low-molecular-weight CD14 of the presentinvention is not specifically limited as far as it contains an antibodythat binds to at least one of human low-molecular-weight CD14 or afragment of the antibody and directly assays the humanlow-molecular-weight CD14 in a specimen. Preferably, it is an assay kitfor human low-molecular-weight CD14 containing the antibody of thepresent invention or a fragment of the antibody as the antibody thatbinds to the human low-molecular-weight CD14 or the fragment of theantibody. More preferably, it is an assay kit for humanlow-molecular-weight CD14 including an antibody prepared using a peptideas an antigen, the peptide having amino acid residues described in anyone of SEQ ID NOS: 2 to 4, or a fragment of the antibody. In addition,preferably, it is an assay kit for human low-molecular-weight CD14including an antibody that binds to a peptide having amino acid residuesdescribed in any one of SEQ ID NOS: 2 to 4 or a fragment of the antibodyas an antibody that binds to the human low-molecular-weight CD14 or afragment of the antibody. Particularly preferably, it is an assay kitfor human low-molecular-weight CD14 including an antibody prepared usinga peptide as an antigen, the peptide having amino acid residuesdescribed in SEQ ID NO: 2 or a fragment of the antibody, or an antibodythat binds to a peptide having 16 amino acid residues described in SEQID NO: 2 or a fragment of the antibody, as an antibody that binds tohuman low-molecular-weight molecule or a fragment of the antibody.

Furthermore, the principle of the assay is not specifically limited asfar as the assay is a method of immunologically assaying humanlow-molecular-weight CD14 using the antibody or the fragment thereof.

As an example of the principle of the assay, an assay kit for humanlow-molecular-weight CD14 (hereinafter, it may be described as asandwich immunoassay kit), which determines the humanlow-molecular-weight CD14 by a sandwich immunoassay using the “antibodythat binds to a peptide having 16 amino acid residues described in SEQID NO: 2” as a preferred example of the antibody according to the secondaspect of the present invention, will be described concretely.

A well-known method may be used as the sandwich immunoassay. Theprinciple, application, and modification of the assay are described in,for example, “Hypersensitive Enzyme Immunoassay”, Eiji Ishikawa Ed.,Center for Academic Publications Japan (1993), “New Utilization Examplesand Applications to Diagnostic Reagent/Drug Development of Immunoassay”,Immunoassay Development Research Society, Keiei-Kyoiku Shuppan, and“Enzyme Immunoassay (3rd Ed), Eiji Ishikawa Ed., Igaku-Shoin Ltd.(1987).

Furthermore, the sandwich immunoassay kit of the present inventioncontains an antibody that binds to a peptide having 16 amino acidresidues described in SEQ ID NO: 2. The characteristic features of theantibody that binds to the peptide having 16 amino acids described inSEQ ID NO: 2, a method of preparing such an antibody, and so on are justas the same as those according to the first aspect of the presentinvention. The antibody may be, but not specifically limited, apolyclonal antibody or a monoclonal antibody.

The sandwich immunoassay is a method using two or more kinds ofantibodies that recognize different sites on a protein to be usuallyassayed, where the assay is performed by forming anantibody-antigen-antibody complex.

First, an insoluble carrier coupled with a first antibody is preparedand is then provided as a solid phase or a reaction place. A specimen isadded to the insoluble carrier provided as the solid phase and then theyare allowed to react with each other. After they have been reacted for apredetermined time period, the solid phase is washed to remove anunbound substance therefrom. Subsequently, a labeled second antibody isadded. After the mixture has been reacted for a predetermined timeperiod, the labeled antibody that did not form a complex is removed bywashing and then the amount of the complex bound to the solid phase isqualitatively or quantitatively determined on the basis of the labeledproduct in a specific manner. The sandwich method may use any one of amethod including two steps as described above (double-step method) and amethod including the step of simultaneously adding both an antigen and alabeled antibody (single-step method).

In the sandwich immunoassay kit of the present invention, the assay isperformed by forming a complex of the “antibody that binds to a peptidehaving 16 amino acid residues described in SEQ ID NO: 2”—humanlow-molecular-weight CD14—the “second binding substance that binds tothe human low-molecular-weight CD14”.

The format of the sandwich immunoassay kit of the present inventionincludes: an insoluble carrier bound with an antibody that binds to apeptide having 16 amino acid residues described in SEQ ID: 2 and asecond binding substance that binds to a labeled low-molecular-weightCD14 (hereinafter, it may be simply described as a second bindingsubstance); or an insoluble carrier bound with a second bindingsubstance and an antibody that binds to a labeled peptide having 16amino acid residues described in SEQ ID NO: 2.

Examples of the second binding substance include an antibody that bindsto the low-molecular-weight CD14. The antibody that binds to thelow-molecular-weight CD14 may be a polyclonal antibody or a monoclonalantibody and is not specifically limited. The monoclonal antibody ispreferable with respect to affinity to the sandwich immunoassay usingthe antibody that binds to the peptide having 16 amino acid residuesdescribed in SEQ ID NO: 2. Furthermore, the second binding substance maybe a fragment of the monoclonal antibody. The fragment of the antibodyis Fab, Fab′, or F(ab′)₂ of the monoclonal antibody.

The antibody that binds to the low-molecular-weight CD14 (hereinafter,it may be described as a second antibody) may be an antibody thatspecifically binds to the low-molecular-weight CD14 or an antibody thatbinds to high-molecular-weight CD14 and is not specifically limited.Preferably, it is an antibody that binds to a site different from thatof the antibody of the present invention. The second antibody is anantibody that binds to a region except a region corresponding to 16amino acids described in SEQ ID NO: 2 of the low-molecular-weight CD14when an antibody that binds to a peptide having 16 amino acid residuesdescribed in SEQ ID NO: 2 is used as the antibody of the presentinvention. More preferably, the second binding substance described aboveis: an antibody that binds to any one region of amino acid residues atpositions 1 to 52 of the human high-molecular-weight CD14 or a fragmentof the antibody; or an antibody competing with or showingcross-reactivity with an antibody that binds to any one region of aminoacid residues at positions 1 to 52 of the human high-molecular-weightCD14 or a fragment of the antibody. Particularly preferably, the secondbinding substance described above is: an antibody that binds to any oneamino acid residue at positions 17 to 26 of the humanlow-molecular-weight CD14 or a fragment of the antibody; or an antibodycompeting with (showing cross-reactivity with) an antibody that binds toany one amino acid residue at positions 17 to 26 of the humanlow-molecular-weight CD14 or a fragment of the antibody.

A polyclonal antibody or monoclonal antibody may be prepared, forexample, using high-molecular-weight CD14, low-molecular-weight CD14, amixture of high-molecular-weight CD14 with low-molecular-weight CD14, orrecombinant CD14 as an antigen, as in the case with the method accordingto the first aspect of the present invention. An exemplified method ofpreparing a second antibody using a mixture of high-molecular-weightCD14 with low-molecular-weight CD14, and recombinant CD14 as antigenswill be shown in Example 3 described below.

In addition, it is preferable to select a second antibody such that,before actually conducting the assay, just as in the case with Example 3described later, a system for the sandwich method including an antibodythat binds to a peptide having 16 amino acid residues described in SEQID NO: 2 and an antibody which is a candidate for the second antibody ispreliminary constructed to confirm the sensitivity of the assay.

Furthermore, the fragments of the antibody: Fab, Fab′, and F(ab′)₂ canbe prepared by a well-known method (“Hypersensitive Enzyme Immunoassay”,written by Eiji Ishikawa, p. 25-40, Center for Academic PublicationsJapan (1993)).

In the sandwich immunoassay, the assay may be performed by a competitionmethod as an alternate of the above method. It is a method of allowingan antigen in a specimen to compete with a labeled antigen or a labeledantigen analogue during the formation of an antibody-antigen-antibodycomplex.

In the sandwich immunoassay kit of the present invention, the assay isperformed by forming a complex of the “antibody that binds to a peptidehaving 16 amino acid residues described in SEQ ID NO: 2”—labeled humanlow-molecular-weight CD14 (or an analogue thereof)—the “second bindingsubstance that binds to human low-molecular-weight CD14”.

The format of the competition method for the sandwich immunoassay kit ofthe present invention includes: an insoluble carrier bounded with anantibody that binds to a peptide having 16 amino acid residues describedin SEQ ID NO: 2; a second binding substance; and labeled humanlow-molecular-weight CD14 or labeled human low-molecular-weight CD14analogue, or includes: an antibody that binds to a peptide having 16amino acid residues described in SEQ ID NO: 2; an insoluble carrierbound with a second binding substance; and labeled humanlow-molecular-weight CD14 or labeled human low-molecular-weight CD14analogue.

Examples of the human low-molecular-weight CD14 analogue include asoluble polypeptide having amino acids at positions 1 to 285 on theN-terminal of the human CD14 (hereinafter, described as sCD14 (1-285))and a recombinant polypeptide having amino acids at positions 1 to 307on the N-terminal of the human CD14 where serine at position 286 issubstituted with cysteine (hereinafter, described as sCD14 (1-307)S286C). In the assay system, however, the human low-molecular-weightCD14 analogue is not specifically limited as far as it is a substancecapable of competing with the human low-molecular-weight CD14 in aspecimen. The methods of preparing sCD14 (1-285) and sCD14 (1-307) S286Care described in WO 01/72993.

Furthermore, in the sandwich immunoassay, the assay may be performed bytaking advantage of the second specific binding as an alternativemethod. It is a method of conducting an assay by forming a complex ofantibody-antigen-antibody—second specific binding substance—specificbinding partner of the second specific binding substance (hereinafter,it may be described as a second specific binding partner).

In the sandwich immunoassay kit of the present invention, the assay isperformed by forming a complex of the “antibody that binds to a peptidehaving 16 amino acid residues described in SEQ ID NO: 2”—humanlow-molecular-weight CD14—the “second binding substance that binds tohuman low-molecular-weight CD14”—second specific bindingsubstance—second specific binding partner, or by forming a complex ofthe “second binding substance that binds to human low-molecular-weightCD14”—human low-molecular-weight CD14—the “antibody that binds to apeptide having 16 amino acid residues described in SEQ ID NO: 2”—secondspecific binding substance—second specific binding partner.

The format taking advantage of the second specific binding of thesandwich immunoassay kit of the present invention includes: an antibodylabeled with a second specific binding substance that binds to a peptidehaving 16 amino acid residues described in SEQ ID NO: 2; a secondspecific binding substance that binds to labeled low-molecular-weightCD14; and an insoluble carrier bound with a second specific bindingpartner, or includes: a labeled antibody that binds to a peptide having16 amino acid residues described in SEQ ID NO: 2; a second bindingsubstance that binds to low-molecular-weight CD14 labeled with a secondspecific binding substance; and an insoluble carrier bound with a secondspecific binding partner.

Examples of the combination of the second specific biding substance andthe second specific binding partner include: an antigen and an antibodythereof; a ligand and a receptor thereof; substance containing somesugars and lectin; and biotin and avidin or streptavidin.

Furthermore, examples of the sandwich immunoassay include: an assay withthe formation of a complex ofantibody-antigen-antibody—anti-immunoglobulin antibody by takingadvantage of an antibody against an antibody, i.e., ananti-immunoglobulin antibody; and an assay with the formation ofanti-immunoglobulin antibody-antibody-antigen-antibody—second specificbinding substance—second specific binding partner by taking advantage ofanti-immunoglobulin antibody and second specific binding.

The sandwich immunoassay kit of the present invention conducts an assayby: forming a complex of the “antibody that binds to a peptide having 16amino acid residues described in SEQ ID NO: 2”—humanlow-molecular-weight CD14—the “second binding substance that binds tohuman low-molecular-weight CD14”—anti-immunoglobulin antibody; forming acomplex of the “antibody that binds to a peptide having 16 amino acidresidues described in SEQ ID NO: 2”—human low-molecular-weight CD14—the“second binding substance that binds to human low-molecular-weightCD14”—anti-immunoglobulin antibody; forming anti-immunoglobulinantibody—the “antibody that binds to a peptide having 16 amino acidresidues described in SEQ ID NO: 2”—human low-molecular-weight CD14—the“second binding substance that binds to human low-molecular-weightCD14”—second specific binding substance—second specific binding partner;forming anti-immunoglobulin antibody—the “second binding substance thatbinds to human low-molecular-weight CD14”—human low-molecular-weightCD14—the “antibody that binds to a peptide having 16 amino acid residuesdescribed in SEQ ID NO: 2”—second specific binding substance—secondspecific binding partner, or the like.

Any sandwich immunoassay is within the scope of the assay of the presentinvention even though a solid phase, a labeled substance, or the like isformed by taking advantage of the second specific binding as far as itperforms an assay by forming a complex of the “antibody that binds to apeptide having 16 amino acid residues described in SEQ ID NO: 2”—humanlow-molecular-weight CD14—the “second binding substance that binds tohuman low-molecular-weight CD14”.

In other words, any sandwich immunoassay kit of the present invention iswithin the scope of the kit of the present invention as far as itincludes an antibody that binds to a peptide having 16 amino acidresidues described in SEQ ID NO: 2.

An insoluble carrier used in the sandwich immunoassay kit of the presentinvention may be beads, latex particles, magnetic particles, a plate, atube, a membrane, or the like. Materials of the beads, plate, or tubeinclude polystyrene, nylon, glass, silicone rubber, stainless steel, andplastic. The membrane may be cellulose, a cellulose derivative,nitrocellulose, a porous synthetic polymer, a glass fiber, cloth, anonwoven fabric, filter paper, or the like. The beads, latex particles,magnetic particles, or the like may be used in a spherical shape. Aspherical shape is advantageous in saving a space in storage. The plateor tube may be used in the form of a well. A well form is advantageousin that it will be accepted to a commercial automatic measuringinstrument, plate reader, or the like. The membrane can be used for animmunochromatographic method or a flow through method described later.

The antibody that binds to a peptide having 16 amino acid residuesdescribed in SEQ ID NO: 2, the second binding substance, the secondspecific binding substance or the partner thereof, or theanti-immunoglobulin antibody can be bound to the insoluble carrier by athermal adsorption method, chemical binding method, or the like.

In addition, it is preferable to subject the non-adsorption surface ofthe insoluble carrier being free of the above substance to a blockingtreatment with any substance that does not affect the assay systembecause the treatment will impart increased specificity or sensitivityto the assay system. The substances that do not affect the assay systeminclude: proteins such as BSA and casein; and surfactants such as Tween20 and NP-40.

Labels to be used in the sandwich immunoassay kit of the presentinvention include: enzymes such as peroxidase, alkali phosphatase,β-_(D)-galactosidase, oxidase, and urokinase; chemiluminescentsubstances such as acridinium or a derivative thereof and aequorin or amodified product thereof; fluorescent substances such as FITC; dyestaff;gold colloid; colored latex; and isotopes.

For instance, in the case of using peroxidase as an enzyme,3,3′,5,5′-tetrabenzidine or 1,2-phenylene diamine may be exemplified asa chromogenic substrate. In the case of using alkali phosphatase,4-nitrophenylphosphate may be exemplified as a chromogenic substrate. Inthe case of using β-_(D)-galactosidase, 2-nitrophenylβ-_(D)-galactosidemay be exemplified as a chromogenic substrate.

Enzyme-labeling to the antibody that binds to a peptide having 16 aminoacid residues described in SEQ ID NO: 2, the second binding substance,the second specific binding substance or the partner thereof, or theanti-immunoglobulin antibody can be performed by a two-stepglutaraldehyde method, periodic acid method, maleimide method, pyridyldisulfide method, or the like.

Apart from the enzyme, a well-known technology such as a thermaladsorption method or chemical binding method may be available in thelabeling.

Enzyme-labeling is preferable because it can be assayed usingconventional chromometry system if any chromogenic substrate exemplifiedabove is used and because the sensitivity thereof is comparatively high.Furthermore, the labeling used in a simple kit such as a kit utilizingan immunochromatographic method or flow through method described lateris preferable because dye stuff, gold colloid, or colored latex can bevisually observed.

The sandwich immunoassay kit of the present invention is characterizedin that an assay is performed by a sandwich immunoassay and includes anantibody that binds to a peptide having 16 amino acid residues describedin SEQ ID NO: 2. The sandwich immunoassay can use the well-knowntechnology as described above. In addition to the above concretedescription, any kit based on the sandwich immunoassay is within thescope of the sandwich immunoassay kit of the present invention and isnot specifically limited as far as the kit includes an antibody thatbinds to a peptide having 16 amino acid residues described in SEQ ID NO:2. In other words, it is enough for the kit to contain an antibody thatbinds to a peptide having 16 amino acid residues described in SEQ ID NO:2 and a reagent required for the sandwich immunoassay. In addition, nocontent is restricted as far as it does not inhibit the assay resultsbased on the principle of the assay.

For instance, a buffer or diluent of a specimen, labeled antibody, orthe like, a chromogenic substrate (see the above description) suitablefor an enzyme when the enzyme is used for a labeled antibody, a blockingagent, a stopping reagent, or a washing solution may be exemplified asan optional constitutional element. In addition, a standard substancemay be also exemplified as an optional constitutional element. Thestandard substances include human low-molecular-weight CD14 and humanlow-molecular-weight CD14 analogues.

Furthermore, a kit that utilizes an immunochromatographic method or aflow through method on the basis of a sandwich immunoassay as aprinciple of the assay is also within the scope of the sandwichimmunoassay kit of the present invention.

The immunochromatographic method is a method where an antigen providedas a test substance in a specimen moves along a test strip to aninsoluble carrier on which an antibody is immobilized while the antigenreacts with a labeled antibody being arranged in the test strip so as tobe able to move, and then a complex of the antibody-antigen-antibody isformed on the insoluble carrier. In general, the antigen can be assayedby a single step of dropping the specimen on the test strip.

For instance, apparatuses for the immunochromatographic method aredisclosed in JP 01-063865 A, WO 88/08534, and WO 90/09592. In addition,apparatuses for the immunochromatographic method having flow channelswith different developing speeds are disclosed in WO 89/03993 and WO99/27364, and for example, the apparatuses can be applied such that alabeled antibody can be reacted after the formation of a complex byallowing the reaction between the immobilized antibody and the antigen.

An example that utilizes the immunochromatographic method of thesandwich immunoassay kit of the present invention will be describedbelow.

For instance, a device (i.e., a kit) is a test strip on which asample-adding part, a reagent part, a detection part, and an absorbingpart are provided such that a liquid specimen added on the sample-addingpart is allowed to move along those parts in that order. It issufficient that a labeled second binding substance be impregnated in thereagent part and an insoluble carrier bound with an antibody that bindsto a peptide having 16 amino acid residues described in SEQ ID NO: 2 bearranged on the detection part.

The specimen added on the sample-adding part absorbs the labeled secondbinding substance at the reagent part. The human low-molecular-weightCD14 reacts with the labeled second binding substance to form a complexwhile they move to the detection part. On the detection part, thecomplex reacts with the antigen that binds to the peptide having 16amino acid residues described in SEQ ID NO: 2, resulting in theformation of a complex of the “antibody that binds to the peptide having16 amino acid residues described in SEQ ID NO: 2”—human low-molecularweight CD14—the “second binding substance that binds to humanlow-molecular-weight CD14” on an insoluble carrier. Any substance andreagent in the specimen, which are not involved in the reaction, move tothe absorbing part. The label of the complex formed on the detectionpart may be determined, particularly may be visually determined.

A porous carrier or the like may be used as a test strip. The porouscarrier may be, for example, nitrocellulose, cellulose, a cellulosederivative, nylon, a nylon fiber, a glass fiber, or a porous syntheticpolymer.

Part of the test strip may be directly used as the sample-adding part orreagent part. Alternatively, for example, cellulose filter paper, aglass fiber, cloth, non-woven fabric, porous synthetic polymer, or thelike may be used depending on the amount of the sample or the dose ofthe reagent.

Cellulose, a cellulose derivative, nitrocellulose, a porous syntheticpolymer, a glass fiber, cloth, non-woven fabric, filter paper, or thelike may be used for the detection part as described above.

A water-absorbable material may be used for the absorbing part. Examplesof the water-absorbable material include: an absorbent polymer such assponge; cellulose filter paper; and filter paper.

The above is one of the examples of the immunochromatographic method. Areference part for confirming the progress of a reaction may be added,or the test strip may be provided with a support or covered with anexternal cover. However, the kit of the present invention is not limitedto them.

Furthermore, as described in the explanation about the sandwichimmunoassay, a kit for an immunochromatographic method, by which acomplex of the “antibody that binds to a peptide having 16 amino acidresidues described in SEQ ID NO: 2”—human low-molecular-weight CD14—the“second binding substance that binds to human low-molecular-weight CD14”is formed on an insoluble carrier and an assay is performed by forming acomplex that utilizes an anti-immunoglobulin antibody and secondspecific binding, is also within the scope of the sandwich immunoassaykit of the present invention.

The flow through method is a method by which an antigen provided as atest substance forms an antibody-antigen-antibody complex together witha solution in a specimen on a membrane provided as an insoluble carrier.At this time, a substance failed to be fixed on the membrane isgenerally removed by perpendicularly passing through the membrane fromthe front to the back.

WO 88/01603 discloses an apparatus based on a multi-step method by whicha specimen, a regent, and a cleaner are dropped onto a membrane.

JP 06-273419 A discloses a method being improved as a single-step methodin which a multi-layered membrane is formed and a regent part isprovided thereon so as to conduct the assay only by dropping a specimen.

Hereinafter, an example of the sandwich immunoassay kit of the presentinvention using a flow through method will be described.

For instance, a device (i.e., a kit) is a kit on which a sample-addingpart, a reagent part, a detection part, and an absorbing part arelayered one on top of another such that a liquid specimen added on thesample-adding part is allowed to move along those parts in that order.It is sufficient that a labeled second binding substance be impregnatedin the reagent part and an insoluble carrier bound with an antibody thatbinds to a peptide having 16 amino acid residues described in SEQ ID NO:2 be arranged on the detection part.

The specimen added on the sample-adding part passes through thesample-adding part perpendicularly from the top to back of the membrane(hereinafter, the same holds true for the sample movement) and thenabsorbs the second binding substance at the reagent part. The humanlow-molecular-weight CD14 reacts with the labeled second bindingsubstance to form a complex while they move to the detection part. Onthe detection part, the complex reacts with the antigen that binds tothe peptide having 16 amino acid residues described in SEQ ID NO: 2,resulting in the formation of a complex of the “antibody that binds tothe peptide having 16 amino acid residues described in SEQ ID NO:2”—human low-molecular weight CD14—the “second binding substance thatbinds to human low-molecular-weight CD14” on an insoluble carrier. Anysubstance and reagent in the specimen, which are not involved in thereaction, move to the absorbing part. The label of the complex formed onthe detection part may be determined, particularly may be visuallydetermined. The label can be visually observed in a simple manner if adevice is designed such that the detection part is detachable from thesample-adding part and reagent part or from the absorbing part. Inaddition, the label can be visually observed from the sample-adding partif each of the sample-adding part and reagent part is made of atranslucent material, or from the lower side if the absorbing part isarranged above the detection part (the sample-adding part side) as inthe case of JP 06-0273419 A.

The same members as those of the immunochromatographic method can beapplied and each member may be formed like a membrane to allow thesolution in a specimen to move.

The above is one of the examples of the flow through method. A referencepart for confirming the progress of a reaction may be added, or eachmember may be provided with a support or covered with an external cover.However, the sandwich immunoassay kit of the present invention is notlimited to them.

Furthermore, as described in the explanation about the sandwichimmunoassay, in addition to the formation of a complex of the “antibodythat binds to a peptide having 16 amino acid residues described in SEQID NO: 2”—human low-molecular-weight CD14—the “second binding substancethat binds to human low-molecular-weight CD14” on an insoluble carrier,a kit for the flow through method that conducts the assay by forming acomplex that utilizes the anti-immunoglobulin antibody and the secondspecific binding is also within the scope of the sandwich immunoassaykit of the present invention.

Furthermore, the sandwich immunoassay kit of the present invention canbe available to an assay based on a MEDIA method (JP 05-264552 A) ofelectrochemically measuring signals from a label and an assay based onan immunoassay method (“Bioscience and Industry”, vol. 61, p. 449-454,2003) using a microchip. The assay kits using those principles arewithin the scope of the sandwich immunoassay kit of the presentinvention as far as they are characterized by their assays based on thesandwich immunoassay and include an antibody that binds to a peptidehaving 16 amino acid residues described in SEQ ID NO: 2.

The sandwich immunoassay kit of the present invention is characterizedby including an antibody that binds to a peptide having 16 amino acidresidues described in SEQ ID NO: 2 and is capable of specificallydetermining low-molecular-weight CD14. A specimen to be used in thesandwich immunoassay kit of the present invention is preferably anaqueous specimen. Particularly preferable examples of the specimeninclude blood, blood component such as serum or plasma, urine or otherbody fluids, cell culture supernatant, and column eluent. They areuseful for the determination on low-molecular-weight CD1 in them.However, from the specimens except the human blood component, such ashuman urine or other body fluids, blood components, urine, or other bodyfluids form species except a human being, cell culture supernatant, andcolumn eluent, proteins, polypeptides, or the like which are analogousto the low-molecular-weight CD14 may be also assayed as well as thelow-molecular-weight CD14. Any assay kit for the above polypeptides, orthe like which are analogous to the low-molecular-weight CD14 is alsowithin the scope of the sandwich immunoassay kit of the presentinvention as far as they each include an antibody that binds to apeptide having 16 amino acid residues described in SEQ ID NO: 2.

Furthermore, in the above explanation, the fragment Fab, Fab′, or(Fab′)₂ of the “antibody that binds to a peptide having 16 amino acidresidues described in SEQ ID NO: 2” may be used instead of the “antibodythat binds to a peptide having 16 amino acid residues described in SEQID NO: 2”.

In the above description, the concrete examples using the “antibody thatbinds to a peptide having 16 amino acid residues described in SEQ ID NO:2” have been described as preferred examples of the antibody accordingto the second aspect of the present invention. However, the antibodyaccording to the first aspect of the present invention, the antibodyaccording to the second aspect of the present invention except the“antibody that binds to a peptide having 16 amino acid residuesdescribed in SEQ ID NO: 2”, or the fragment Fab, Fab′, or (Fab′)₂ ofthose antibodies may be also used.

Preferable is a sandwich immunoassay kit using the antibody of thesecond aspect of the present invention. More preferable is a sandwichimmunoassay kit using the “antibody that binds to a peptide having 16amino acid residues described in SEQ ID NO: 2”.

Furthermore, principles for the assay include an agglutination method,solid-phase binding method, and solution reaction method in addition tothe sandwich immunoassay method. Depending on those methods, therespective kits may be constructed such that each of them contains theantibody that binds to at least one of human low-molecular-weight CD14or the fragment of the antibody, preferably the antibody of the presentinvention or the fragment of the antibody.

In the agglutination method, the antibody is bound on the surface ofparticles and the presence of the antigen cause the particles toagglutinate, so that the antigen can be qualitatively or quantitativelydetermined in a specific manner in reference to the degree ofagglutination of the particles.

An agglutination immunoassay kit of the present invention conducts theassay by forming the “antibody of the present invention”—humanlow-molecular-weight CD14 and causing the agglutination thereof.

The format of the agglutination immunoassay kit of the present inventionincludes particles to the surface of which the antibody of the presentinvention binds.

The particles used may be those generally used, including latex, redblood cells (e.g., sheep red blood cells), gelatin, micro beads, carbonparticles, or the like.

The solid-phase binding method is a method of conducting the assay bythe formation of a complex between an antibody and an antigen on a solidphase. An antigen-containing specimen is adsorbed in an insolublecarrier (i.e., solid phase, the same shall apply hereinafter). Next, alabeled antibody is added and the mixture is reacted to qualitatively orquantitatively determine the amount of the complex bound on the solidphase in a specific manner on the basis of the labeled product.

Furthermore, as a competition method, an antigen analogue is adsorbed onan insoluble carrier to allow the labeled antigen to compete with thereaction with the antigen in the specimen to determine the amount of thelabeled antibody bound to the antigen analogue. Furthermore, as analternative method of the competition method, the antibody is adsorbedin the insoluble carrier and the reaction with the antigen in thespecimen is competed with the labeled antigen analogue to determine theamount of the labeled antigen analogue bound to the antibody.

In the solid-phase binding immunoassay kit of the present invention, anassay is performed by forming an “antibody of the presentinvention”—human low-molecular-weight CD14 complex, an “antibody of thepresent invention”—labeled human low-molecular-weight CD14 (or ananalogue thereof) complex, or a “labeled antibody of the presentinvention”—human low-molecular-weight CD14 (or an analogue thereof)complex.

The format example of the solid-phase binding immunoassay kit of thepresent invention: includes the antibody of the present invention, aninsoluble carrier, and a reagent for adsorbing a specimen on theinsoluble carrier; or

includes the antibody of the present invention and an insoluble carrierbound with labeled human low-molecular-weight CD14 (or an analoguethereof); or

includes an insoluble carrier bound with the labeled antibody of thepresent invention and an insoluble carrier bound with labeled humanlow-molecular-weight CD14 (or an analogue thereof).

The insoluble carrier, human low-molecular-weight CD14 analogue, andlabeling and adsorbing regents are the same as those described in theexplanation of the sandwich immunoassay kit.

A solution-reaction method may be a method of qualitatively orquantitatively determining low-molecular-weight CD14 in a specificmanner by: making a reaction between an antigen and a labeled antibodyin a liquid phase; and then separating an antigen-antibody complex fromthe antigen and antibody by a coagulation process with the antibody orby physical and chemical procedures.

The format example of the solution-reaction immunoassay kit performs anassay such that a complex of the “labeled antibody of the presentinvention”—human low-molecular-weight CD14 is formed in a liquid phaseand then an unbound labeled antibody is removed therefrom.

The format example of the solution-reaction immunoassay kit of thepresent invention includes the labeled antibody of the presentinvention.

In addition, in the above explanation, the “fragment of the antibody ofthe present invention, Fab, Fab′, or (Fab′)₂” may be used instead of the“antibody” of the present invention.

The examples of the assay kit of the present invention have beendescribed above on the basis of their assay principles. However, the kitof the present invention is not limited to those principles. As far asan assay kit contains an antibody that binds to at least one humanlow-molecular-weight CD14 or a fragment of the antigen, the assay kit iswithin the scope of the assay kit of the present invention. For theprinciples of the immunoassay, the technologies well known in the artare available. “Hypersensitive Enzyme Immunoassay”, Eiji Ishikawa Ed.,Center for Academic Publications Japan (1993), “New Utilization Examplesand Applications to Diagnostic Reagent/Drug Development of Immunoassay”,Immunoassay Development Research Society, Keiei-Kyoiku Shuppan, and“Enzyme Immunoassay (3rd Ed), Eiji Ishikawa Ed., Igaku-Shoin Ltd.(1987), which are mentioned above, may be also referred.

The level of low-molecular-weight CD14 which can be specificallydetermined by the kit of the present invention increases in a patientsuffering from sepsis. Thus, the assay of low-molecular-weight CD14 willbe provided as a diagnostic index of sepsis and the kit of the presentinvention is useful for diagnosis of sepsis.

According to a fourth aspect of the present invention, there is providedan assay method for low-molecular weight CD14 with which the assay ofhuman low-molecular-weight CD14 in a specimen is directly conductedusing an antibody that binds to at least one of humanlow-molecular-weight CD14 for detecting the human low-molecular-weightCD14 without detecting human high-molecular-weight CD14.

The assay method of the present invention is a method for the assay ofhuman low-molecular-weight CD14 for detecting the humanlow-molecular-weight CD14 without detecting human high-molecular-weightCD14 and uses an antibody that binds to at least of one of the humanlow-molecular-weight CD14 to directly determine the humanlow-molecular-weight CD14 in a specimen. Preferably, it is a method forthe assay of low-molecular-weight CD14 using the antibody of the presentinvention. More preferably, it is a method for the assay oflow-molecular-weight CD14 using an antibody prepared using a peptide asan antigen, the peptide having amino acid residues described in any oneof SEQ ID NOS: 2 to 4. In addition, it is preferably a method for theassay of low-molecular-weight CD14 using an antibody that binds to apeptide having amino acid residues described in any one of SEQ ID NOS: 2to 4. It is particularly preferably a method for the assay oflow-molecular-weight CD14 using an antibody prepared using a peptide asan antigen, the peptide having amino acid residues described in SEQ IDNO: 2, or using an antibody that binds to a peptide having 16 amino acidsequences described in SEQ ID NO: 2. In the above explanation,furthermore, the “fragment of the antibody, Fab, Fab′, or (Fab′)₂” maybe used instead of the “antibody”.

Preferably, furthermore, it is a method for a low-molecular-weight CD14assay with which human low-molecular-weight CD14 is determined by asandwich immunoassay.

The antibody of the present invention may be used as an immobilizedantibody, labeled antibody, or the like. In addition, a method for theassay that utilizes second specific binding and an anti-immunoglobulinantibody is also included. In this case, the antibody of the firstaspect of the present invention can be used as a free antibody, anantibody binding to a second specific binding substance or to a secondspecific binding partner, or the like.

The method for the assay of the present invention may be anon-competitive or competitive method of sandwich immunoassay, and alsothe measurement with an immunochromatographic method or a flow-throughmethod may be included.

Furthermore, the principle of the assay method of the present inventionis not limited to the sandwich immunoassay and other examples thereofinclude an agglutination method, a solid-phase binding method, and asolution reaction method.

The details are as described in the third aspect of the presentinvention.

According to a fifth aspect of the present invention, there is provideda diagnostic method for sepsis by which human low-molecular-weight CD14is directly assayed.

The diagnostic method for sepsis directly assays the low molecularweight CD14.

The method of directly assaying the low-molecular-weight CD14 is asdescribed in the fourth aspect of the present invention. Furthermore,the diagnosis can be performed using the kit described in the thirdaspect of the present invention.

As described in Examples 3, 10, and 11 below, the assay of thelow-molecular-weight CD14 in blood from each of normal individuals andvarious kinds of patients confirmed that a patient suffering from sepsisspecifically showed a high level of low-molecular-weight CD14. This factmeans that the result obtained by the assay using the above kit can beused as an index in the diagnosis of sepsis. For instance, the level oflow-molecular-weight CD14 in blood of a patient is determined and isthen compared with the standard level of normal individuals obtained,for example, by averaging up their measurements, or with the range ofthe levels of the normal individual. For instance, the average+2SD or3SD of normal individuals is used as a cut-off level and, when the levelof low-molecular-weight CD14 is higher than such a level, it is definedas a positive index. In addition, an index for the diagnosis can be alsoprovided by comparing the measured level of low-molecular-weight CD14 ofeach individual with the levels of low-molecular-weight CD14 of normalindividuals and patients suffering from sepsis or the standard levelsobtained by standardizing those levels in advance. For instance, thelow-molecular-weight CD14 level of a normal individual is defined as of0 to 0.1 μg/ml and the level of a patient suffering from sepsis isdefined as of 0.2 μg/mL or more, followed by comparing with the measuredlevel to provide a negative, pseudo positive, or positive index.

According to a sixth aspect of the present invention, there is provideda peptide having amino acid residues described in any of SEQ ID NOS: 2to 4. The peptide of the present invention consists of amino acidresidues described in any one of SEQ ID NOS: 2 to 4. The peptide of thepresent invention is useful as an antigen for preparation of theantibody of the present invention.

According to a seventh aspect of the present invention, there isprovided a method of preparing the antibody of the present invention, inwhich an antigen is a peptide having consecutive 8 to 30 amino acidresidues selected from the amino acid sequence described in SEQ IDNO. 1. Preferable examples of the peptide used as the antigen includethe peptide of the sixth aspect of the present invention and a peptidehaving consecutive 8 or more amino acids of the amino acid residuesdescribed in SEQ ID NO: 2. The “peptide having consecutive 8 or more and16 or less amino acids of the amino acid residues described in SEQ IDNO: 2” means a peptide that contains any one of the following (1) to(9), which is a sequence on the upstream and/or downstream side followedafter the sequence described below in SEQ ID NO: 2 and preferablyconsists of 10 or more, 12 or more, or 16 or more amino acids in total.

1) Arg Val Asp Ala Asp Ala Asp Pro (SEQ ID NO: 6) 2) Val Asp Ala Asp AlaAsp Pro Arg (SEQ ID NO: 7) 3) Asp Ala Asp Ala Asp Pro Arg Gln (SEQ IDNO: 8) 4) Ala Asp Ala Asp Pro Arg Gln Tyr (SEQ ID NO: 9) 5) Asp Ala AspPro Arg Gln Tyr Ala (SEQ ID NO: 10) 6) Ala Asp Pro Arg Gln Tyr Ala Asp(SEQ ID NO: 11) 7) Asp Pro Arg Gln Tyr Ala Asp Thr (SEQ ID NO: 12) 8)Pro Arg Gln Tyr Ala Asp Thr Val (SEQ ID NO: 13) 9) Arg Gln Tyr Ala AspThr Val Lys (SEQ ID NO: 14)

The details of the method of the present invention are as described inthe section for the aspect of the antibody of the present invention.

The peptide of the present invention can be prepared by the methoddescribed in the section for the aspect of the antibody of the presentinvention.

EXAMPLES

Hereinafter, the present invention will be described more concretely byway of examples. However, the examples are only exemplary and thepresent invention should by no means be construed as being limitedthereto. Further, symbols used in the following description are based onthe symbols as a convention in the art.

Those manufactured by ProMedDx and Sera Care Life Science were purchasedand used as sera of normal individuals and sera of patients sufferingfrom sepsis used in the following examples.

Example 1 Preparation of Polyclonal Antibody Using Synthetic Peptide asAntigen

1-(1) Preparation of Peptide as Antigen <1>

To bind a peptide having the sequence described in SEQ ID NO: 2(corresponding to a sequence at positions 53 to 68 described in SEQ IDNO: 5) (hereinafter, described as S68 peptide) to a carrier protein atthe N-terminal thereof through an SH group, the peptide was synthesizedby inserting cysteine into the N-terminal. That is, using a peptidesynthesizer ABI433A (Applied), amino acid columns were aligned accordingto the amino acid sequence and an amino acid column for cysteine wasplaced on the N-terminal, followed by conducting automatic synthesis.The synthesized peptide was cut out from a resin by a conventionalprocedure and was then precipitated with ether, recovered, and dissolvedin distilled water again, followed by freeze drying. After the resultingcrude peptide had been dissolved, the peptide was eluted with a lineargradient of 5-70% acetonitrile concentration using a C18 reverse phaseHPLC (CAPCELL-PAK, Shiseido Corp.), followed by collecting a fractioncontaining a target peptide. The collected fraction was freeze-dried and2 to 3 mg of purified peptide was obtained.

1-(2) Preparation of Peptide Carrier Antigen Using Synthetic Peptide <1>

Each of two kinds of peptides prepared in 1-(1) was dissolved indistilled water to 10 mg/mL and the solution was mixed with 10 mg/mL ofmaleimide-activated keyhole limpet hemocyanin (Imject MaleimideActivated Mariculture Keyhole Limpet Hemocyanin (KLH) (PIERCE)) inequivalent amounts. After the mixture had been reacted for 2 hours atroom temperature, the reaction mixture was desalted by an NAP-10 column(Amersham Bioscience) being equilibrated with physiological saline toobtain 1 mg of S68-peptide carrier antigen (hereinafter, described asS68 peptide-KLH). The concentration of proteins described in thefollowing examples was obtained by dividing the amount of used KLH bythe amount of liquid.

1-(3) Preparation of Peptide as Antigen <2>

Two kinds of peptide sequences represented in Table 1 were synthesizedusing a peptide synthesizer (PSSH-8, Shimadzu Corporation) by the sameway as that of 1-(1) and purified, respectively. Each of the obtainedpeptides was about 5 mg in amount. By the way, the “number” in the tablerepresents the name of a peptide explained below and the “position”represents the position thereof found in the amino acid sequencedescribed in SEQ ID NO: 5.

TABLE 1 SEQ Number Position Amino acid sequence ID NO P001  1-17 Thr ThrPro Glu Pro Cys 3 Glu Leu Asp Asp Glu Asp Phe Arg Cys Val Cys P002 14-32Arg Cys Val Cys Asn Phe 4 Ser Glu Pro Gln Pro Asp Trp Ser Glu Ala PheGln Cys1-(4) Preparation of Peptide Carrier Antigen Using Synthetic Peptide <2>

Each of the peptides prepared in 1-(3) was dissolved in PBS (pH 7.2)containing 0.1 M EDTA, and as in the case of 1-(2) 3 mg of each ofpeptide carrier antigens where KLH bound to the respective peptides wasobtained.

1-(5) Preparation of Polyclonal Antibody Using Synthetic Peptide <1>

For preparing a polyclonal antibody against S68 peptide-KLH prepared in1-(2), a rabbit was immunized using S68 peptide-KLH. That is, 100 μg ofeach of S68 peptide-KLH was diluted with 500 μL of physiological salineand the solution was mixed with 500 μL of Freund's complete adjuvant(DIFCO) in equivalent amounts, followed by subcutaneously administeringthe mixture to the back of New Zealand white female rabbit (KitayamaLabes) weighing 2.1 to 2.2 kg. After 2 weeks, 100 μg of each of S68peptide-KLH was diluted with 500 μL of physiological saline and thesolution was mixed with 500 μL of Freund's incomplete adjuvant (DIFCO)in equivalent amounts, followed by subcutaneously administering themixture to the back. After additional 2 weeks from that, 100 μg of S68peptide-KLH was diluted with 1 mL of physiological saline and thesolution was administered in an ear vein.

After 1 week from the completion of administration, blood was collectedfrom the ear vein and antiserum was separated from the blood by routineprocedures and an antibody was purified. First, ammonium sulfate wasadded to the antiserum up to a final saturation concentration of 33%.After the mixture had been stirred for 1 hour at 4° C., the separatedprecipitate was centrifuged. Then, the precipitate was dissolved in a76-mM phosphate buffer (hereinafter, described as PBS (pH 6.4)) and thesolution was dialyzed overnight. After the dialysate had been filtered,the filtrate was applied to a protein A column (Prosep-A, Millipore).Then, a binding IgG fraction was eluted with a 0.1 M glycinehydrochloride buffer (pH 3.0) to obtain a purified antibody. Afterdialysis with PBS (pH 6.4), the protein concentration was calculatedfrom the absorbance at a wavelength of 280 nm (absorption coefficient:0.533 mg/mL). Hereinafter, the obtained antibody will be described as anS68 peptide polyclonal antibody.

1-(6) Preparation of Polyclonal Antibody Using Synthetic Peptide asAntigen <2>

Using each of the peptide carrier antigens prepared in 1-(4), as in thecase of 1-(3), the immunization and the purification of antiserum wereperformed to prepare each of peptide polyclonal antibodies (P001 andP002 polyclonal antibodies). Furthermore, the immunization was performedsuch that the peptide carrier antigen (0.5 mg/rabbit) was administered 5times in two months. After the whole blood had been collected, each ofthe antisera (antiserum P001 and P002) was obtained.

1-(7) Preparation of Specific Purified Polyclonal Antibody

For purifying only an antibody against S68 peptide from the S68-peptidepolyclonal antibodies, specific purification was performed by thefollowing method. First, for biding the S68 peptide inserted withcysteine (hereinafter, described as C-S68 peptide) to a carrier throughan SH group, 200 μg of C-S68 peptide was mixed per 1 mL of SufoLinkCoupling Gel (PIERCE) and reacted according to the manual thereof. Afterthe completion of the reaction, the remaining active group was blockedand then an S68 peptide-biding affinity column was prepared. Next, 7.92mg of the purified IgG fraction described in 1-(3) was applied and thenthe column was washed with a phosphate buffer (pH 7.4) (Dulbecco,hereinafter, described as D-PBS (pH 7.4)), followed by eluting ananti-S68-peptide antibody with 0.1 M glycine hydrochloride buffer (pH3.0). After the elution, pH was readjusted to neutral and then dialysiswas performed with PBS, followed by calculating the proteinconcentration from an absorbance at 280 nm (absorption coefficient:0.533 mg/mL). As a result, 0.52 mg of an anti-S68-peptide antibody(hereinafter, described as S68 antibody) was obtained.

Example 2 Preparation of Monoclonal Antibody Using Synthetic Peptide asAntigen

20 μg of S68 peptide-KLH prepared in Example 1-(2) was dissolved in 100μL of physiological saline and mixed with an equivalent amount ofFreund's complete adjuvant (DIFCO), followed by administering 100 μL ofthe mixture to each of the rear foot pads of a female Wister rat aged 8weeks. After 2 weeks, the iliac lymph node was surgically excised andcell fusion was performed. The cell fusion was conducted according toTamie Ando and Takeshi Chiba: “Introduction to Monoclonal AntibodyExperimental Manipulation”, page 83, 1991 (Kodansha). In other words,lymphocytes were separated from the lymph node using a cell strainer(Falcon) and mixed with myeloma cells (Sp2/O-Ag14) at a ratio of 5:1,followed by cell fusion using polyethylene glycol. Fused cells weresuspended in an HAT medium and hybridomas were selected, followed byscreening hybridomas producing the target antibody.

The screening was performed by an ELISA method in which sCD14 (1-307)S286C was directly immobilized on a plate. That is, 50 μL of sCD14(1-307) S286C diluted with 0.1-M phosphate buffer (pH 7.4) to 1 μg/mLwas added to each well of an immunoplate (Maxisorb, NUNC) and left tostand for 1 hour at 37° C. After that, the plate was washed withion-exchanged water 5 times and then 100 μL of PBS (pH 6.4) containing0.1% BSA was added to each well, followed by leaving the plate instanding for 1 hour at room temperature to effect blocking. Then, theculture supernatant sampled from the selected hybridomas was added toeach well and allowed to react at 37° C. for 1 hour. After that, theplate was washed 3 times with physiological saline containing 0.05%Tween 20. Subsequently, 50 μL of a solution obtained by dilutingperoxidase-labeled anti-rat immunoglobulin antibody (DAKO) with PBScontaining 10% rabbit serum 1000-fold was added to each well. Afterreaction at 37° C. for 1 hour, the plate was washed 5 times in the samemanner as above and a tetramethylbenzidine solution (TMB, BioFix) wasadded to each well. After a reaction for 10 minutes at room temperature,the reaction was stopped with a 0.5 M sulfuric acid solution and anabsorbance at 450 nm was measured using a plate spectrophotometer(NJ-2100, Japan Intermed). As a result, a well containing hybridomacapable of producing an antibody binding to sCD14 (1-307) S286C wasselected.

Next, from the selected well, cloning was performed by a limitingdilution method according to Tamie Ando and Takeshi Chiba: “Introductionto Monoclonal Antibody Experimental Manipulation”, page 83, 1991(Kodansha). After 10 days, likewise, screening was performed using as anindex the reactivity with sCD14 (1-307) S286C and 6 kinds of hybridomaswere selected. The selected hybridomas were cultivated in a 10%FCS/RPMI1640 medium (Sigma) and then cultivated in Hybridoma-SFM medium(Invitrogen) to produce an antibody. The antibody was purified using aprotein G column (Prosep-G column, Millipore). The subtype of thepurified F1146-17-2 antibody was determined to be rat IgG2b·κ by using arat typing kit (ZYMED).

By the way, sCD14 (1-307) S286C was prepared using the method describedin Example 9 of WO 01/72993.

Example 3 Study of Assay System for Human Low-Molecular-Weight CD14

Using the antibodies described in Examples 1 and 2, the assay system forhuman low-molecular-weight CD14 with a sandwich EIA method was studied.

3-(1) Preparation of Recombinant Human CD14

First, for preparing a monoclonal antibody against sCD14 (1-285) to beused as a second antibody in the sandwich ELISA method, sCD14 (1-285) asan immunogen was prepared in E. coli. In order to express sCD14 (1-285)in E. coli, an expression plasmid pTrp1659 was constructed by thefollowing method.

First, oligomer 8, links (5′-agc tta gga att t-3′) (SEQ ID NO: 15) andoligomer 8, linkA (5′-cta gaa att cct a-3′) (SEQ ID NO: 16) weresynthesized.

Those oligomers were mixed in equivalent amounts and heated at 99° C.for 1 minute, and the mixture was then annealed by gradually cooling itdown to room temperature. Furthermore, 5′-terminal thereof wasphosphorylated by T4 Polynucleotide Kinase to prepare a linker.

Next, sense primer (5′-aca tct aga tga cca cgc cag aac ct-3′) (SEQ IDNO: 17) and antisense primer (5′-ttt gga tcc tta cta gag atc gag cactct-3′) (SEQ ID NO: 18) were synthesized and PCR was performed usingPyrobest DNA Polymerase and plasmid pM1659 described in Example 8 of WO01/72993 as a template.

After a reaction solution had been heated for 2 minutes at 90° C., thecycle of 98° C. for 10 seconds, 55° C. for 30 seconds, and 72° C. for 1minute was repeated 30 times.

The resulting amplified product of about 90° bp was double-digested withXbaI and BamHI to collect DNA fragments. The vector pM710 described inExample 10 of JP 06-025289 A was double-digested with HindIII and BamHIand then subjected to agarose gel electrophoresis and collected. Afterthree-ligation of the linker already phosphorylated, PCR-amplified DNAfragment/XbaI+BamHI digested fragment, and vector/HindIII+BamHIfragment, which were described above, the resultant was transformed intoE. coli competent cells (JM109 (TOYOBO) to obtain a clone containing thetarget plasmid. Plasmid DNA was prepared by routine procedures.

Subsequently, JE7924 transformant strain for the production of sCD14(1-285) was prepared using an electroporation method.

First, E. coli JE7924 (J. Bacteriol 173, p. 4799, (1991)) was restoredfrom a glycerol stock and incubated in an LB medium at 37° C. overnight.Furthermore, the bacteria were inoculated to 50 ml of a fresh LB mediumand continuously incubated until the absorbance at 600 nm reached 0.5 to0.6, followed by directly ice-cooling a culture flask for 30 minutes.Next, E. coli cells were collected and washed twice with ice-cooledsterilized distilled water and once with an ice-cooled 10% glycerolsolution, followed by being suspended in 100 μL of an ice-cooled 10%glycerol solution. The suspension was dispensed into two tubes with 50μL aliquots and quickly frozen in liquid nitrogen to prepare competentcells (JE7924), which were saved at −80° C. until the time of use.

Next, 50 μL of JE7924 competent cells was transformed with 30 ng of byelectroporation device, Gene Pulser of BIO-RAD Co., Ltd. In addition,the settings at this time were a voltage of 2.5 kV and a resistance of200 Ω, and a capacitance of 25 μF. After that, the resultant wasincubated in an LB agar plate containing 50 μg/mL of ampicillinovernight to obtain a clone transformed with pTrp1659. The clone thereofwas incubated at 37° C. overnight in an LB medium and was theninoculated into a fresh medium, followed by being incubated foradditional 5 hours. OD at 600 nm of culture suspension reached to 2 to3, 3β-indole acrylic acid (Sigma CO., Ltd.) was added in a finalconcentration of 100 μg/mL and the mixture was incubated at 37° C. for 4hours, resulting in induction expression of sCD14 (1-285). Next, E. coliwas collected and then an inclusion body was prepared using Bug BusterProtein Extraction Reagent (Novagen, Co., Ltd.). After that, theinclusion body was dissolved in an SDS-PAGE buffer and an SDS-PAGE wascarried out to identify the expression of sCD14 (1-285) by Westernblotting by an anti-CD14 antibody.

Similarly, sCD14 (1-285) to be used as an immunogen was prepared byincubating a JE7924 transformant strain in 1 L of an LB medium. First,the culture solution was centrifuged. After E. coli cells had beencollected, the bacteria cells were washed with D-PBS and 50 mL of BugBuster Protein Extraction Reagent (Novagen, hereinafter described as BugBuster) was added to the collected bacteria cells. The bacterial cellswere suspended and left standing for 30 minutes at room temperature.After lysing, the bacterial cells were subjected to a 10-minutesonication treatment (US-3, Iuchi Seieido) and centrifuged at 10000×g at4° C. for 20 minutes to remove a supernatant. Likewise, an additionalsonication treatment was performed on the cells and the resultingprecipitate was suspended in 50 mL of Bug Buster. The suspension wasadded with 1 mL of a 10-mg/mL lysozyme (Seikagaku Corporation), and thewhole was gently stirred and left standing for 10 minutes at roomtemperature. Subsequently, 200 mL of 1/10 volume of high-concentrationBug Buster was added to the mixture and the whole was stirred, followedby being subjected to centrifugation similarly to remove a supernatant.The resulting precipitate was suspended by the addition of 200 mL of1/10 concentration of Bug buster and then the suspension was centrifugedsimilarly, followed by repeating such an operation several times. 100 mLof D-PBS was added in the finally obtained precipitate, resulting in aninclusion body.

For the preparation of sCD14 (1-285), the inclusion body was dissolvedin a TE buffer (pH 8.0, Nippon Gene) containing 1% Triton-X100 and thesolution was then subjected to freeze and thawing 3 times, following bycollecting a precipitate by centrifugation. The precipitate wasdissolved in the TE buffer (pH 8.0, Nippon Gene) containing 1%Triton-X100 again, and the solution was ice-cooled and then subjected toa 12-minute ultrasonic treatment with 250 μA at intervals of 10 secondsand centrifuged, followed by collecting a precipitate. The precipitatewas dissolved in a TE buffer (pH 8.0, Nippon Gene) containing 1%Triton-X100 and 0.2M NaOH, and then treated at 37° C. for 10 minutes,centrifuged, and re-dissolved three times, followed by collecting aprecipitate. The resulting precipitate was dissolved in an aqueoussolution containing 6 M guanidine hydrochloric acid to prepare purifiedsCD14 (1-285). The concentration thereof was calculated by a proteinassay of Bradford using BSA as a standard preparation.

3-(2) Preparation of Anti-CD14 Monoclonal Antibody

[1] Preparation of F1106-13-3 Antibody

Using sCD14 (1-285) derived from E. coli described above as an antigento be administered, a monoclonal antibody was prepared. First, 20 μg ofpurified sCD14 (1-285) was mixed with Freund's complete adjuvant (DIFCO)in equivalent amounts, followed by intraabdominally administering 200 μLof the mixture to a 6-week-old female ddy mouse. After 2 weeks, 20 μg ofpurified sCD14 (1-285) was mixed with Freund's incomplete adjuvant(DIFCO) in equivalent amounts, followed by intraabdominallyadministering 200 μL of the mixture. 50 μL of antigen wasintraabdominally administered to the mouse 3 days before cell fusion.After 3 days, spleen was aseptically excised. Lymphocytes were isolatedfrom the spleen and mixed with myeloma cells (P3×63-Ag. 8. U.1) in aratio of 10:1 and fusion was performed with polyethylene glycolaccording to a method described on Tamie Ando and Takeshi Chiba:“Introduction to Monoclonal Antibody Experimental Manipulation”, page83, 1991 (Kodansha). After hybridomas had been selected using an HATmedium, screening of hybridomas producing antibodies biding to sCD14(1-285) was performed by an ELISA method.

First, sCD14 (1-285) was diluted with PBS (pH 6.4) to 0.4 μg/mL and 50μL of the resultant solution was then added to each well of animmunoplate (Maxisorb, NUNC) and reacted at 4° C. overnight. After that,the plate was washed with ion-exchanged water 5 times and then 100 μL ofPBS (pH 6.4) containing 0.5% BSA was added to each well for blocking.Then, the sampled culture supernatant was added to each well and allowedto react at 37° C. for 1 hour. After that, the plate was washed 3 timeswith physiological saline containing 0.05% Tween 20. Subsequently, 50 μLof a solution obtained by diluting peroxidase-labeled anti-mouseimmunoglobulin antibody (DAKO) with PBS containing 10% rabbit serum1000-fold was added to each well. After a reaction at 37° C. for 1 hour,the plate was washed 5 times in the same manner as above and atetramethylbenzidine solution (TMB, BioFix) was added to each well.After a reaction for 10 minutes at room temperature, the reaction wasstopped with a 0.5 M sulfuric acid solution and an absorbance at 450 nmwas measured using a plate spectrophotometer (NJ-2100, Japan Intermed).On the basis of the result, a well containing hybridoma producing anantibody binding to sCD14 (1-285) was selected. Next, from the selectedwell, cloning was performed by a limiting dilution method according toTamie Ando and Takeshi Chiba: “Introduction to Monoclonal AntibodyExperimental Manipulation”, page 83, 1991 (Kodansha). After 10 days,likewise, screening was performed using the reactivity with sCD14(1-285) as an index to select hybridomas. As a result, 12 types ofhybridomas producing anti-sCD14 (1-285) monoclonal antibody wereselected.

The selected hybridomas were cultivated in a 10% FCS/RPMI1640 medium(Sigma) and then cultivated in Hybridoma-SFM medium (Invitrogen) toproduce an antibody. The antibody was purified using a protein A column(Prosep-A, Millipore).

The subtype of F1106-13-3 antibody, which was an antibody having aparticularly high sensitivity, was determined as IgG2b·κ using IsoStripMouse Monoclonal antibody Isotyping Kit (Roche).

(2) Preparation of F1031-8-3 Antibody

F1031-8-3 antibody was prepared using the method described in Example 7of WO 01/22085. Briefly describing, 20 μg of CD14 protein derived fromin human blood was dissolved in physiological saline and the solutionwas mixed with Freund's complete adjuvant (DIFCO) in equivalent amounts.Then, after 1 week from each of the initial intraabdominaladministration and the second thereof 2 weeks after the initial, anincreased level of antibody titer in serum was confirmed by an ELISAmethod on the reactivity with recombinant human CD14 protein as in thecase of Example 5 of WO 01/22085. A 100-μg antigen was intraabdominallyadministered to a mouse as a final administration and after 3 days thespleen was surgically excised from the mouse. Lymphocytes were isolatedfrom the spleen and mixed with myeloma cells (P3×63-Ag. 8. U.1) in aratio of 10:1 and cell fusion was performed with polyethylene glycol.Hybridomas were selected using an HAT medium and after one weekscreening of hybridomas producing antibodies was performed by the ELISAmethod described above. The hybridoma that had reacted with theimmobilized soluble CD14 protein was cloned by a limiting dilutionmethod. After 10 days, similarly, screening was performed to obtain ananti-CD14 monoclonal antibody. F1031-8-3 antibody having the subtype ofIgG2b·κ determined using IsoStrip Mouse Monoclonal antibody IsotypingKit (Roche) was obtained as a typical antibody.

3-(3) Study of Assay System for Human Low-Molecular-Weight CD14

For preparing a system capable of specifically detecting humanlow-molecular-weight CD14, a sandwich EIA system was prepared using theantibodies described in Examples 1, 2, and 3-(2).

[1] Preparation of Peroxidase-Labeled Antibody

A peroxidase-labeled antibody was prepared according to the method ofNakane et al. (J. Histochem. Cytochem., vol. 22, p. 1084, 1974). Thatis, 4 mg of peroxidase (Toyobo) was dissolved in distilled water and thesolution was then reacted at 25° C. for 20 minutes by the addition of100 mM of periodic acid. After the completion of the reaction, 1.5%ethylene glycol was added to the reaction product and the whole wasreacted at 25° C. for 10 minutes, followed by dialyzing against a 1-mMacetate buffer (pH 4.4). Each of the purified F1031-8-3 antibody andF1106-13-3 antibody was dialyzed with a 10-mM bicarbonate buffer (pH9.5), and then 4 mg of peroxidase activated by the addition of 70 μL ofa 0.2-M bicarbonate buffer (pH 9.5) per 4 mg was mixed with the antigenin equivalent amounts to allow a reaction at 25° C. for 2 hours. Next, 4mg/mL of sodium borohydride was added and then the reaction wascontinued for additional 2 hours at 4° C. The reaction solution wasdialyzed with PBS, resulting in a peroxidase-labeled F1031-8-3 antibody(hereinafter, it may be described as F1031-8-3-HRP) andperoxidase-labeled F1106-13-3 antibody (hereinafter, it may be describedas F1106-13-3-HRP). The concentration of antibody was calculated fromthe amount of antibody used and the volume of the labeled antibodysolution.

[2] Preparation of Sandwich EIA System <1>

Prepared was a 2-step sandwich EIA system using the S68 antibodyprepared as an immobilized antibody in Example 1 and antibodies preparedin Example 3-(2)[1] and [2] as labeled antibodies. That is, S68 antibodywas diluted with D-PBS (pH 7.4) to 10 μg/mL and 50 μL of the resultantsolution was then added to each well of an immunoplate (Maxisorb, NUNC)and reacted at 4° C. overnight. After that, the plate was washed withion-exchanged water 5 times and then 100 μL of D-PBS containing 0.1%StabilGuard (SurModics, Inc) and 0.1% Tween 20 was added to each well toeffect blocking. Using as a diluent PBS (pH 7.4) containing 1% normalindividual serum (serum from which soluble CD14 was removed using 3C10,hereinafter, described as CD14—absorbing serum) and 0.1% BSA, dilutedspecimens of human sera of normal individuals and human sera of patientssuffering from sepsis were prepared by diluting the sera 20-fold,respectively. A diluted specimen was added in a concentration of 50 μLper well and reacted at 37° C. for 2 hours.

After the completion of the reaction, the specimen was washed threetimes with physiological saline containing 0.05% Tween 20 and 50 μL ofF1031-8-3-HRP or F1106-13-3-HRP diluted to 0.6 μg/mL with 76 mM PBS (pH8.0) containing 5% rat serum, 1% mouse serum and 0.1% Tween 20 was addedto each well. After a reaction at 37° C. for 2 hours, the plate waswashed 5 times in the same manner as above and a tetramethylbenzidinesolution (TMB, BioFix) was added to each well. After a reaction for 20minutes at room temperature, the reaction was stopped with a 0.5 Msulfuric acid solution and an absorbance at 450 nm was measured using aplate spectrophotometer (NJ-2100, Japan Intermed). As a result, as shownin Table 2, a soluble protein in blood, i.e., the low-molecular-weightCD14 being defined in the present invention, which could not increase ina normal individual but increase in a patient suffering from sepsis inthe system in which antibody derived from S68 peptide was used, was ableto be assayed.

[3] Preparation of Sandwich EIA System <2>

1) Prepared was a 2-step sandwich EIA system using the F1146-17-2antibody prepared as an immobilized antibody in Example 2 antibodyprepared in Example 3-(2) and [2] as a labeled antibody. F1146-17-2antibody was diluted with PBS (pH 6.4) to 120 μg/mL and 50 μL of theresultant solution was then added to each well of an immunoplate(Maxisorb, NUNC) and reacted at 56° C. for 30 minutes. After that, theplate was washed with ion-exchanged water 5 times and then 100 μL of PBScontaining 0.1% StabilGuard (SurModics, Inc) and 0.1% Tween 20 (WakoPure Chemical Industries, Ltd.) was added to each well to effectblocking. Using as a diluent PBS (pH 6.4) containing 1% BSA, dilutedspecimens of human sera of normal individuals and human sera of patientssuffering from sepsis were prepared by diluting the sera 10-fold,respectively. A diluted specimen was added in a concentration of 50 μLper well and reacted at 25° C. for 2 hours.

After the completion of the reaction, the plate was washed three timeswith physiological saline containing 0.05% Tween 20 and 50 μL ofperoxidase-labeled F1031-8-3 antibody diluted to 0.5 μg/mL by 76 mMphosphate buffer (pH 8.0) containing 5% rat serum, 1% mouse serum, and0.1% Tween 20 was added to each well. After a reaction at 25° C. for 2hours, the plate was washed 5 times in the same manner as above and atetramethylbenzidine solution (TMB, BioFix) was added to each well.After a reaction for 20 minutes at room temperature, the reaction wasstopped with a 0.5 M sulfuric acid solution and an absorbance at 450 nmwas measured using a plate spectrophotometer (NJ-2100, Japan Intermed).As a result, similarly to the S68 antibody, in the case of S68-peptidespecific monoclonal antibody as shown in Table 2, low-molecular-weightCD14, which was almost not found in the sera of normal individuals butfound in a high level in the sera of patients suffering from sepsis, wasable to be assayed. That is, the result confirmed that an antibody thatbinds to S68 peptide can prepare a sandwich system irrespective ofwhether the antibody is polyclonal or monoclonal.

2) A two-step sandwich EIA system, where an immobilized antibody usedwas the polyclonal antibody prepared using the synthetic peptide as anantigen in Example 1-(6), was prepared. An assay was conducted using asspecimens sera of human normal individuals and human patients sufferingfrom sepsis by the same way as that of 3-[2], but P001 polyclonalantibody, P002 polyclonal antibody, or P012 polyclonal antibody was usedin place of S68 antibody. As a result, as shown in Table 2, similarly tothe S68 antibody, in the case of the polyclonal antibody using thesynthetic peptide as an antigen, low-molecular-weight CD14, which wasalmost not found in the serum of a human normal individual but found ina high level in the serum of a patient suffering from sepsis, was ableto be assayed. The results confirmed that a sandwich system can beperformed even in a system using an antibody prepared using a peptide asan antigen, the peptide having 8 to 16 amino acid residues selected fromthe amino acid sequences at positions 1 to 285 of humanhigh-molecular-weight CD14.

In Table 2, “++” represents a 4-fold or more absorbance at 450 nmcompared with the absorbance of the diluent itself and “+” represents a2-fold or more absorbance, and “−” represents an absorbance equal to theabsorbance of the diluent.

TABLE 2 Measured level Combination of antibodies Patient ImmobilizingLabeling suffering from Normal side side sepsis individual S68 antibodyF1031-8-3 ++ − antibody S68 antibody F1106-13-3 ++ − antibody F1146-17-2F1031-8-3 + − antibody antibody P001 F1031-8-3 + − polyclonal antibodyantibody P002 F1031-8-3 + − polyclonal antibody antibody[4] Preparation of Sandwich EIA System <3>

A 3-step sandwich EIA system using F-1031-8-3 antibody as an immobilizedantibody and 368 antibody as a labeled antibody was prepared. Thepresent EIA system was performed by biotinylating the S68 antibody asfollows. That is, 50 μL of D-Biotinoyl-ε-Aminocaproic AcidN-Hydroxysuccinimide Ester (Roche) prepared to 300 μg/mL by dissolvingin DMSO was added to 0.5 mL of S68 antibody prepared to a concentrationof 0.93 mg/mL by substituting with a 0.05-M phosphate buffer (pH 8.0)containing 0.15 M NaCl and the mixture was reacted while being stirredfor 2 hours at room temperature. After the completion of the reaction,the reaction product was substituted with PBS (pH 7.4) by a desaltingcolumn (NAP-5, Amersham Bioscience). The concentration of the preparedbiotinylated S68 antibody (hereinafter, it may be described as Bio-S68antibody) was calculated using an absorption coefficient of 1.4 on thebasis of absorbance at 280 nm.

The sandwich EIA system immobilized F1031-8-3 antibody on an immunoplate(NUNC) and blocked. A blocking solution was removed. Then, 500 ng/mL ofsCD14 (1-307) s286c (hereinafter, it may be described as a standardpreparation) dissolved in 0.1% BSA/PBS and a solution with no standardpreparation added were added to wells as negative control, respectively.The plate was washed after a reaction at 37° C. for 1 hour, andsubsequently 50 μL of biotinylated S68 antibody prepared to 1 μg/mL bydiluting with PBS (pH 7.4) containing 2% rat serum, 1% mouse serum, 1%rabbit serum, and 0.1% Tween 20 was added and reacted at 37° C. for 1hour. After the completion of the reaction, the plate was washed andthen a 10,000-fold diluted peroxidase-labeled streptavidin (which may bedescribed as SA-HRP, Invitrogen) was added. The plate was washed after areaction for 1 hour. After a color had been developed with a TMBsolution (BioFix), the reaction was terminated by a terminating liquidand an absorbance at 450 nm was measured using a plate spectrophotometerE-Max (Molecular Device, Co., Ltd.).

As shown in Table 3, in the present system, a sandwich EIA system wasable to be prepared. In other words, the inventors confirmed that asandwich assay system can be prepared even if an antibody that binds toS68 peptide is used as an immobilized antibody or used as a freeantibody or labeled antibody. In Table 3, “++” represents that theabsorbance difference with 0 to 500 ng/mL of the standard preparation is0.5 Abs or more, “+” represents 0.1 or more, and “−” represents lessthan 0.1.

[5] Preparation of Sandwich EIA System <4>

A 1-step EIA system was prepared such that immobilized and labeledantibodies were of the same system as that of [2], and a specimen andthe labeled antibody were simultaneously added. That is, 25 μL of eachof 0- and 500-ng/mL standard preparations was added to aS68-antibody-immobilized plate, followed by the addition of 25 μL ofF1031-8-3-HRP prepared to 1 μg/mL by dilution with PBS (pH 7.4)containing 2% rat serum, 1% mouse serum, 1% rabbit serum, and 0.1% Tween20. A reaction was carried out for 1 hour at 37 C°. After the completionof the reaction, the plate was washed and colored by a TMB solution(BioFix). Then the reaction was terminated by a terminating liquid,followed by measuring an absorbance at 450 nm using a plate absorbancemeter E-Max (Molecular Device, Co., Ltd.). As shown in Table 3, asandwich EIA system was also made in the present system. That is, theinventors confirmed that a sandwich assay system using an antibody thatbinds to S68 peptide can conduct an assay without any relation to thereaction sequence.

[6] Preparation of Sandwich EIA System <5>

Immobilized and labeled antibodies were of the same system as that of[2], and a specimen and the labeled antibody were simultaneouslyreacted. Then, a 2-step EIA system to react with the immobilizedantibody was prepared. That is, 25 μL of each of 0- and 500-ng/mLstandard preparations was mixed with 25 μL of F1031-8-3-HRP prepared to2 μg/mL with PBS (pH 7.4) containing 2% rat serum, 1% mouse serum, 1%rabbit serum, and 0.1% Tween 20. After the completion of the reaction,the reaction solution was added to an S68-antibody-immobilized plate,and the whole was reacted at 37C.° for 1 hour. The plate was washed andthen colored by a TMB solution (BioFix) and then the reaction wasterminated by a terminating liquid, followed by measuring an absorbanceat 450 nm using a plate absorbance meter E-Max (Molecular Device, Co.,Ltd.). As shown in Table 3, a sandwich EIA system was also made in thepresent system. That is, the inventors confirmed that a sandwich assaysystem using an antibody that binds to S68 peptide can conduct the assaywithout any relation to the reaction sequence.

[7] Preparation of Sandwich EIA System <6>

A sandwich EIA system using the specific biding of biotin-streptavidinwas prepared.

1) Assay system using streptavidin on the immobilizing side

Streptavidin (PIERCE) diluted to 10 μg/mL with PBS (pH 7.4) wasdispensed into immunoplates (NUNC) with 50 μL aliquots and immobilizedby treating it at 4° C. overnight, respectively. After blocking, theliquid was discarded from them and 25 μL of each of biotinylated S68antibodies prepared to 2 μg/mL with PBS (pH7.4) containing 2% rat serum,1% mouse serum, 1% rabbit serum, and 0.1% Tween 20 and 0- and 500-ng/mLstandard preparations dissolved in 0.1% BSA/PBS was added. After areaction for 1 hour at 37° C., the plate was washed and subsequently 50μL of F1031-8-3-HRP diluted to 1 μg/mL was added, followed by a reactionat 37° C. for 1 hour. After the completion of the reaction, the platewas washed and colored by a TMB solution (BioFix) and then the reactionwas terminated by a terminating liquid, followed by measuring anabsorbance at 450 nm using a plate spectrophotometer E-Max (MolecularDevice, Co., Ltd.). The present systems were tested similarly even ifthe standard preparation, biotinated S68 antibody, andperoxidase-labeled F1031-8-3 antibody were simultaneously added. Asshown in Table 3, sandwich EIA systems were able to prepared in bothsystems.

2) Assay system using peroxidase-labeled streptavidin

The present system was prepared by the method shown in [4]. Furthermore,a 2-step method was studied, where a reaction was carried out at 37° C.for 1 hour after simultaneous addition of a standard preparation andbiotinated F1031-8-3 antibody (which may be described as Bio-F1031-8-3)prepared according to [4] and about 10,000-fold dilutedperoxidase-labeled streptavidin (Invitrogen) was added after washing.After the completion of the reaction, the plate was washed and coloredby a TMB solution (BioFix) and then the reaction was terminated by aterminating liquid, followed by measuring an absorbance at 450 nm usinga plate spectrophotometer E-Max (Molecular Device, Co., Ltd.). As shownin Table 3, in the present system, a sandwich EIA system was able bealso prepared. That is, the inventors confirmed that the assay can beattained even if an immobilized or labeled substance is prepared using asecond specific binding such as the biding of biotin and streptavidin asfar as low-molecular-weight CD14 is sandwiched between an antibody thatbinds to S68 peptide and an antibody that binds to the assay analyte,low-molecular-weight CD14 in human serum. By the way, “Str” representsstreptavidin and “Bio” represents biotinylating.

TABLE 3 Step Example Plate 1 2 3 Reactivity [4] F1031-8-3 StandardBio-S68 SA- + antibody prepa- antibody HRP ration [5] S68 Standard − −++ antibody prepa- ration F1031-8-3- HRP [6] S68 Standard S68 − ++antibody prepa- antibody ration Plate F1031-8-3- HRP [7] (1) Str Bio-S68F1031-8-3- − + antibody HRP Standard preparation [7] (1) Str Bio-S68 − −++ antibody Standard prepa- ration F1031-8-3- HRP [7] (2) S68 StandardSA-HRP − ++ antibody prepa- ration Bio-F1031-8-3

Example 4 Preparation of Immunochromatographic Assay System

4-(1) Immunochromatographic Method Using Gold-Colloid Labeled Antibody<1>

An assay system which could be easily used in a laboratory or by bedsidewas prepared. The outline of the assay system was shown in FIG. 1(A).First, a gold colloid-labeled F1106-13-3 antibody was prepared by mixing1 mL of gold colloid (40 nm in particle diameter, B. B. International)with 9 μg of F1106-13-3 antibody. Next, a conjugate pad was prepared.That is, the gold colloid-labeled F1106-13-3 antibody was diluted with aconjugate-applying buffer so that an absorbance at 520 nm would be about1.5 and 1 mL of the resultant solution was then applied on a 33-Glassstrip of 10×150 nm, followed by drying under reduced pressure overnight.At this time, the antibody titer of gold colloid-labeled F1106-13-3antibody in a reagent per test was about 50 units (1 unit equals 1 μL ofgold colloid-labeled F1106-13-3 antibody at OD520=1.0). Theantibody-immobilized membrane was prepared as follows. S68 antibody wasdiluted to 1 mg/mL with PBS (pH 7.4) and the solution was linearlyapplied on a nitrocellulose membrane (FF85/100, Schleicher & Schuell) inan amount of 0.75 μL/cm using an inkjet coating machine manufactured byBioDot Co., Ltd. At this time, a control line (anti-mouse polyclonalantibody, DAKO) was simultaneously applied. After drying, the membranewas immersed in a blocking liquid containing 0.5% casein for 30 minutesand then an excess part of the liquid was removed, followed by dryingagain. Next, an immunochromatographic reagent was formulated using eachof the prepared materials. That is, a conjugate pad, immobilizedmembrane, an upper-absorbing pad (#900 filter paper, Schleicher &Schuell), or sample-dropping pad (33-Glass glass fiber filter,Schleicher & Schuell) was attached on a PB020 plastic-backing sheet(BioDot) and then cut in 5 mm in width by a strip cutter manufactured byBioDot Co., Ltd. The cut strip was housed in a housing case (NIPPNTechnocluster, Inc.) and provided as an immunochromatographic reagent.

An assay was performed as described below using the prepared reagent. Astandard preparation diluted 10″ folds within the range of 10,000 to 1ng/mL with 1% BSA-PBS was provided as a sample. Then, 100 μL of thesample was dropped into the reagent to determine the presence or absenceof a line after the mixture had been left to stand at room temperaturefor 20 minutes. The criteria for the judgment were as follows:

(++): Level at which a thick line is developed so that the line can beclearly judged as positive;

(+): Level at which color development can be judged as a line eventhough the color development is pale;

(±): Level at which what looks like color development is slightlyobserved but difficult to be recognized as a line; and

(−): Level at which no color development is recognized.

As a result, as shown in FIG. 2 and Table 4, the sensitivity of “+” ormore was obtained at a sample concentration of 10 ng/mL or more.Therefore, the result confirmed that the assay can be performed simplyand quickly by an immunochromatographic system.

4-(2) Immunochromatographic Method Using Gold Colloid-Labeled Antibody<2>

The assay was conducted while the immobilized antigen and goldcolloid-labeled antibody of the immunochromatographic assay systemprepared in 4-(1) were arranged inversely. The gold-colloid marker ofS68 antibody and immunochromatographic system were able to be preparedby the same way as that of 4-(1). As a result, as shown in Table 4, thesensitivity of “+” or more was obtained at a sample concentration of 100ng/mL or more.

TABLE 4 Gold colloid- Sample concentration (ng/mL) labeled Immobilized10000 1000 100 10 1 0 F1106-13-3 S68 ++ ++ + + ± − S68 F1106-13-3 ++ + +± ± −4-(3) Preparation of Immunochromatographic Method UsingStreptavidin-Biotin System

In addition, an immunochromatographic assay using a streptavidin-biotinsystem was prepared. The outline of the assay was shown in FIG. 1(B).First, according to Example 3-2[4], F1031-8-3 antibody was biotinated.Then, a gold colloid-labeled streptavidin was prepared by mixing 1 mL ofgold colloid (40 nm in particle diameter, B. B. International) with 10μg of streptavidin. The gold colloid-labeled streptavidin was dilutedwith a conjugate-applying buffer so that an absorbance at 520 nm wouldbe about 1.5 and 1 mL of the resultant solution was then applied on a33-Glass strip of 10×150 nm, followed by drying under reduced pressureovernight. At this time, the antibody titer of gold colloid-labeledstreptavidin in a reagent per test was about 50 units (1 unit equals 1μL of gold colloid-labeled streptavidin at OD520=1.0). Theantibody-immobilized membrane was prepared as follows. S68 antibody wasdiluted to 1 mg/mL with PBS (pH 7.4) and the solution was linearlyapplied on a nitrocellulose membrane (FF85/100, Schleicher & Schuell) inan amount of 0.75 μL/cm using an inkjet coating machine manufactured byBioDot Co., Ltd. At this time, a control line (anti-mouse polyclonalantibody, DAKO) was simultaneously applied. After drying, the membranewas immersed in a blocking liquid containing 0.5% casein for 30 minutesand then an excess part of the liquid was removed, followed by dryingagain. Next, an immunochromatographic reagent was formulated using eachof the prepared materials.

That is, a conjugate pad, immobilized membrane, an upper-absorbing pad(#900 filter, Schleicher & Schuell), or sample-dropping pad (33-Glassglass fiber filter, Schleicher & Schuell) was attached on a PB020plastic-backing sheet (BioDot) and then cut in 5 mm in width by a stripcutter manufactured by BioDot Co., Ltd. The cut strip was housed in ahousing case (NIPPN Technocluster, Inc.) and provided as animmunochromatographic reagent. An assay was performed as described belowusing the prepared reagent. A standard preparation diluted 10″ foldswithin the range of 10,000 to 1 ng/mL with 1% BSA-PSS was provided as asample. Then, 100 μL of the sample was dropped into 100 μL of reagentcontaining 0.1 μg of biotinized F1031-8-3, and the whole was mixed.Then, 100 μL of the mixture was dropped into a sample-dropping pad ofthe housing case to determine the presence or absence of a line afterthe mixture had been left to stand at room temperature for 20 minutes.Therefore, in the present system, the sensitivity of “+” or more wasalso obtained at a concentration of 100 ng/mL or more just as in thecase of (1).

Example 5 Preparation of Flow-Through Assay System

A flow-through assay system is prepared according to JP 06-273419 A.That is, 1 g of a disperse dye (Red Violet, Kayaron, Co., Ltd.) issuspended in 10 mL of distilled water, and then resuspended in 5 ml ofdistilled water after being washed with distilled water. 0.2 mL of0.5-mg/mL F1031-8-3 antibody diluted with physiological saline is addedto 0.2 mL of the disperse dye and the whole is incubated at 45° C. for30 minutes. After the resultant has been cooled on ice, centrifugalseparation is performed. The resulting precipitate is resuspended in PBS(pH 7.4) containing 0.5% BSA and 10% lactose to prepare a dispersedye-labeled F1031-8-3 antibody. Next, the disperse dye-labeled F1031-8-3antibody is dispensed with 0.1 mL aliquots and immersed into filterpaper (No. 63, Advantec Toyo) cut into 14 mm in diameter, followed byfreeze-drying to prepare a porous body adhered with a soluble reagent.

Immobilization on a membrane is performed as follows. First, 2 mg/mL ofS68 antibody diluted with physiological saline is applied on anitrocellulose membrane (Advantec Toyo) of 5 microns in pore diameterand dried at 37° C. Next, blocking is performed using PBS (pH 7.4)containing 1% BSA to prepare an antibody-immobilized membrane. Theprepared materials are assembled in a housing case in the followingorder. An assay reagent is prepared by assembling the porous bodyadhered with a soluble reagent, antibody-immobilized membrane,polypropylene-laminated filter paper (No. 28, Advantec Toyo), and atransparent plate made of polycarbonate of 0.5 mm in thickness in order.An assay is initiated by the addition of 0.5 mL of a sample to the assayreagent and a judgment is performed by observing color from the backside by the naked eyes after the sample has been completely absorbed.

Example 6 Specificity of S68 Antibody

For confirming the specificity of S68 antibody prepared in Example 1,the inventors studied whether blocking occurs by a peptide by the sameassay as that of Example 3-(3). That is, S68 peptide (amino acidsequence at positions 53 to 68), synthetic polypeptide prepared by thesame way as that of Example 1 (amino acid sequence at positions 53 to58, amino acid sequence at positions 57 to 62, and amino acid sequenceat positions 59 to 64), or negative control peptide (SEQ ID NO: 19)(CysGlu Gly Asn Gly Asn Asn Phe Glu Ser Arg Glu Ala Cys) was diluted to 0,0.1, 1, and 10 μg/mL and 25 μL of each diluted solution was added to 25μL Of each of 50-fold diluted solutions of the sera obtained frompatients suffering from sepsis and the sera of normal individuals toinitiate a competitive reaction by mixing with S68 antibody. After that,the levels of low-molecular-weight CD14 bound to S68 antibody withoutinhibition by any peptide were determined. As a result, as shown in FIG.3, in both the sera of the normal individuals showing low levels and ofpatients suffering form sepsis showing high levels, the binding betweenS68 antibody and the low-molecular-weight protein in blood was inhibitedin the case of S68 peptide but not inhibited in the case of otherpartial peptides (each containing 6 amino acids) and a negative controlpeptide. The above result confirmed that a protein being detected inblood by S68 antibody is specifically recognized by S68 antibody. Inaddition, the result also confirmed that the sequence recognized by theantibody requires a length of at least 7 amino acids because theinhibition can not be attained by three kinds of synthetic peptides (thenumber of amino acids: 6) corresponding to the partial peptides of S68peptide.

Example 7 Reaction Rate Constant of Prepared Antibody

The specificities and reaction rate constants of S68 antibody preparedin Example 1 and F1146-17-2 antibody prepared in Example 2 were analyzedusing Biacore 3000 (Biacore), respectively. First, S68 peptide-BSA to beimmobilized was prepared by the same way as one described in Example 1using maleimidated BSA (Imject Maleimed Activated BSA, PIERCE). Next,the S68 peptide-BSA was immobilized on a censor tip CM5 (Biacore) usingan amine-coupling kit (Biacore). An assay was performed such that HBS-EP(Biacore) was used as a running buffer and a dilution series (50, 100,150, 200, and 300 nM) of F1146-17-2 antibody was injected into flowcells. The data analysis was performed using Biaevaluation softwareversion 3.0 (Biacore) by subtracting reference-cell data from flow-cellmeasurement data of S68 peptide-BSA. As a result of analyzing adissociation constant (KD), the F1146-17-2 antibody showed affinity ashigh as 4.8×10⁻⁹ M. By the way, the KD value of specifically-purifiedrabbit S68 peptide polyclonal antibody measured similarly was 2.2×10⁻¹⁰M.

Example 8 Specificity of Anti-CD14 Monoclonal Antibody

8-(1) Analysis of F1106-13-3 Antibody

For clarifying a binding region (epitope) of F1106-13-3 antibody, apeptide library membrane (Custom SPOTs, Sigma Genosys) on which theamino acid sequence of CD14 was synthesized from the N-terminal thereof10 amino acids at a time was used for analysis. That is, the membranewas blocked based on the instruction manual thereof and then was reactedwith F1106-13-3 antibody, washed, and then reacted withβ-galactosidase-bound anti-mouse antibody. After the membrane had beenwashed, a peptide sequence on which the antibody was bound was detectedusing X-gal. By the way, the peptide sequences on the peptide librarymembrane were analyzed using 19 peptides which were synthesized suchthat 10 amino acids were subjected to the synthesis at a time so as tooverlap two amino acids of the respective C terminals of the sequencesof amino acids at positions 1 to 154. The peptides were prepared by thesame way as that of Example 1-(1).

The result found that F1106-13-3 antibody binds to the regioncorresponding to an amino acid sequence at positions 17 to 26 of SEQ IDNO: 5(CNFSEPQPDW) from the N-terminal of high-molecular-weight CD14.

8-(2) Analysis of F1031-8-3 Antibody <1>

For confirming the specificity of F1031-8-3 antibody, using sCD14(1-285) derived from E. coli described in Example 3-(1) and sCD14(1-356) and sCD14 (1-307) S286C prepared from COS cells using methodsdescribed in Examples 8 and 9 of WO 01/72993, the binding activity wasdetermined.

First, sCD14 (1-356), sCD14 (1-307) S286C, sCD14 (1-285), or BSA wasimmobilized 250 ng/spot on a membrane, Hybond-C extra (AmershamBioscience), and after drying it was blocked by 0.05% Tween 20containing 0.05 g/mL of skim milk (Meiji Milk Products), PBS (pH 6.4).After the resultant had been left to stand for 1 hour at roomtemperature, F1031-8-3 antibody diluted to 3 μg/mL with 0.05% Tween 20containing 0.5% BSA, PBS (pH 6.4) was added and reacted for 1 hour atroom temperature, followed by washing with 0.05% Tween 20, PBS (pH 6.4).

Next, peroxidase-labeled anti-mouse immunoglobulin antibody (DAKO)diluted 500 folds with 0.05% Tween 20 containing 10% rabbit serum, PBS(pH 6.4) was added and reacted for 30 minutes at 37° C. Then, themembrane was washed similarly, followed by confirming the bindingactivity of the antibody with ECL kit (Amersham Bioscience). As aresult, as shown in Table 5, F1031-8-3 antibody bound to sCD14 (1-285),sCD14 (1-307) S286C, and sCD14 (1-356) derived from E. coli not to BSA.Thus, the result found that the F1031-8-3 antibody specificallyrecognized all types of CD14 proteins. In Table 5, “+” represents asituation in which a spot was detected on a film and “−” represents asituation in which no spot was detected.

TABLE 5 sCD14 sCD14 (1-307) sCD14 (1-356) S286C (1-285) BSA Bindingactivity + + + −

8-(3) Analysis of F1031-8-3 Antibody <2>

For clarifying a binding region (epitope) of F1031-8-3 antibody, thespots analysis was performed as in the case of 8-(1). However, in thespots method, no recognition region of F1031-8-3 antibody could bespecified. For the purpose of analyzing the similarity of therecognition regions of both antibodies, in the sandwich EIA system ofExample 3-(3) ([2] where S68 antibody was used as immobilized one andF1031-8-3-HRP was used as labeled one, an inhibition test was performedusing F1106-1-3 antibody.

First, as in the case of Example 3-(3)[2], 100 ng/mL of the standardpreparation was added to and reacted with an S68-antibody-immobilizedplate. After the plate had been washed, before the addition ofF1031-8-3-HRP, a 25-μL buffer containing 6 μg/mL of F1106-13-3 antibody,mouse IgG antibody, or no antibody was added. Then, 25 μL ofF1031-8-3-HRP antibody was added, followed by the measurement by thesame way as that of Example 3-(3)-[2].

As shown in Table 6, no inhibition occurred in the mouse IgG antibodyaddition system while the inhibition of binding between F1031-8-3 andstandard preparation by F1106-13-3 antibody occurred. This factindicated that F1106-13-3 antibody may bind to at least one region to berecognized by F1031-8-3 antibody. By the way, an “inhibition rate” wascalculated from each absorbance being decreased at the time of definingthe absorbance of the buffer alone as 100%.

TABLE 6 Additive Inhibition rate (%) Mouse IgG antibody 2 F1106-13-3antibody 70

Example 8 Assay Kit for Human Low-Molecular-CD14

8-(1) Typical Format of Assay Kit for Sandwich EIA System

A typical format of a soluble protein kit using a combination ofimmobilized and labeled antibodies that show high levels of humanlow-molecular-CD14 in the specimen patients suffering from sepsis andlow levels in specimen from normal individuals in Example 3-(3) will bedescribed below.

-   <1> Immobilized antibody: Plate on which S68 antibody is immobilized-   <2> Labeled antibody: Peroxidase-labeled F1031-8-3 antibody-   <3> Substrate solution (tetramethylbenzidine solution)-   Other Accessories-   Configuration Example of a Plate System-   <4> Plate-washing solution (0.9% NaCl, 0.05% Tween 20 solution>-   <5> Sample-diluting solution (0.1%-BSA-containing PBS solution)<-   <6> Reaction-terminating liquid (0.5 M H₂SO₄ solution)-   <7> Standard preparation (CD14 (1-307) S286C)

Measuring Instruments for Performing an Assay Using the Above Assay Kit<Example>

-   <8> Plate spectrophotometer (e.g., E-Max (Molecular Device, Co.,    Ltd.))    8-(2) to (11) Configuration Examples of Assay Kit for Sandwich EIA    System

In addition to 8-(1), the examples of the assay kit for a sandwich EIAsystem are shown in Table 7. <1> represents a binding substanceimmobilized on a plate. <2> represents a labeled binding substance. Theconstituent elements of <3> to <7> and a measuring instrument <8> as areference example are identical with 8-(1). <9> represents an antibodybound with a second specific biding substance.

TABLE 7 <1> <2> <9> (2) F1146-17-2 F1031-8-3-HRP antibody (3)S68antibody F1106-13-3-HRP (4) F1146-17-2 F1106-13-3-HRP antibody (5)F1031-8-3 S68 antibody- antibody HRP (6) F1031-8-3 F1146-17-2-HRPantibody (7) F1106-13-3 S68 antibody- antibody HRP (8) F1106-13-3F1146-17-2-HRP antibody (9) F1031-8-3 SA-HRP Bio-S68 antibody antibody(10)  Str F1031-8-3-HRP Bio-S68 antibody (11)  S68 antibody SA-HRPBio-F1031-8-38-(12) Standard Curve of Assay Kit for Sandwich EIA System

Using the assay kit of (1), an assay was performed by the same way asthat of Example 3-(3)[2]. That is, S68 antibody was diluted to 10 μg/mLwith D-PBS (pH 7.4) and 50 μL of the resultant solution was then addedto each well of an immunoplate (Maxisorb, NUNC). After a reaction at 4°C. overnight, the plate was washed five times with ion-exchanged waterand blocked by the addition of 100 μL of D-PBS containing 0.1%StabilGuard (SurModics, Inc.) and 0.1% Tween 20 to each well. Next, 76mM PBS (pH 7.4) containing 1% CD14-absorbing serum and 0.1% BSA was usedas a diluent to prepare a dilution series of 0, 3, 25, 60, 100, and 150ng/mL of CD14 (1-307) S286C protein standard preparation. The dilutionseries of the standard preparation was added in an amount of 50 μL perwell and reacted for 2 hours at 37° C. After the completion of thereaction, the plate was washed three times with physiological salinecontaining 0.05% Tween 20. Then, 50 μL of diluted labeled antibodiesprepared by diluting 5% rat serum, 1% mouse serum, andperoxidase-labeled F1031-8-3 antibody to 0.6 μg/mL with 76 mM PBS (pH8.0) containing 0.1% Tween 20 were added to each well. After a reactionat 37° C. for 2 hours, the plate was washed five times in the same wayas above and a tetramethylbenzidine solution (TMB, BioFix) was added toeach well. After a reaction for 20 minutes at room temperature, thereaction was terminated by a 0.5 M sulfuric acid solution and anabsorbance at 450 nm was measured using a plate spectrophotometer(NJ-2100, Japan Intermed). A standard curve prepared was shown in FIG.4. A simple assay system with high sensitivity as a measuringsensitivity of 0.6 ng/mL (blank+3SD) was realized.

8-(13) Specificity of Sandwich EIA System

For studying the influence of high-molecular-weight CD14 present inhuman serum on the assay system prepared, soluble CD14 derived fromnormal individual serum at a concentration of 0 to 4 μg/mL was added tothe standard preparation of CD14 (1-307) S286C to conduct the same assayas that of (12). As a result, there was no influence on the measuredlevel even though the concentration of the soluble CD14 derived fromnormal individual serum was 4 μg/mL. The result found that thecross-reactivity of the present sandwich EIA system withhigh-molecular-weight CD14 was 0.3% or less. In other words, the resultconfirmed that the present system does not detect human serumhigh-molecular-weight CD14 and is specific to a soluble protein showinga high level in serum of a patient suffering from sepsis.

8-(14) Evaluation on Assay Kit for Sandwich EIA System

Reproducibility of the assay results of the kit of (1) was evaluated.The coefficient of variation (CV) of within-run reproducibility using 3samples of specimens as in the case of (12) was 5.8, 3.6, and 3.5% andreproducibility between measurements was 6.2, 5.2, and 5.1%,respectively. Thus good results were obtained, while no influence of ananticoagulant (heparin, citric acid, or EDTA) was observed. The resultsdescribed above showed that the present kit has a sufficient ability forthe assay of human low-molecular-weight CD14.

8-(15) Example of Immunochromatographic Assay Kit

-   <1> Labeled antibody: F1031-8-3 antibody labeled with gold colloid-   <2> Conjugate pad: Glass fiber filter (33-Glass strip, manufactured    by Schleicher & Schuell) on which <1> is applied-   <3> Antibody-immobilized membrane: Nitrocellulose membrane    (FF85/100, manufactured by Schleicher & Schuell) blocked by 0.5%    casein and having an immobilizing line of S68 antibody and a control    line (an immobilizing line of anti-mouse polyclonal antibody) on the    downstream of the immobilizing line.-   <4> Sample-dropping pad: 33-Glass glass fiber filter (manufactured    by Schleicher & Schuell)<-   <5> Absorbing pad (#900 filter paper (manufactured by Schleicher &    Schuell)-   <6> Sheet: PB020 plastic backing sheet (manufactured by BioDot); <2>    to <5> are assembled on <6> such that a liquid dropped in <4> is    allowed to flow through <2>, <3>, and <5> in this order.-   <7> Housing case (OEM case available from NIPPN Technocluster, Inc.)

By the way, the outlines of <1> to <5> are represented in FIG. 1(A).

8-(16) to (19) Example of Immunochromatographic Assay Kit

Table 8 shows, in addition to 8-(15), examples of an assay kit for asandwich EIA system utilizing the second specific binding betweenbinding of biotin and streptavidin, and examples of an assay kit for asandwich EIA system utilizing the fragment of an antibody that binds toa peptide having 16 amino acid residues described in SEQ ID NO: 2. <1>represents a labeled binding substance. The constituent elements <2> to<7> are identical. As a substance to be applied on <3>, <3>-(i)represents a binding substance to be immobilized on an immobilizedmembrane and <3>-(ii) represents a binding substance to be immobilizedon a control line. <8> represents an antibody bound with a secondspecific binding substance, the substance being a reagent to be appliedon <2> or <4> as in the case of <1>, or to be added to a specimen orsimultaneously added together with the specimen.

By the way, the outlines of (16)<1> to <5> are shown in FIG. 1(B), and(17) to (20) are similarly understood.

TABLE 8 <1> <3>-(i) <3>-(ii) <8> (16) Gold colloid- S68 Anti-mouseBio-F1031-8-3 labeled Str antibody polyclonal antibody antibody (17)Gold colloid- S68 Str Str-F1031-8-3 labeled Bio antibody antibody (18)Gold colloid- Str Anti-mouse Bio-S68 labeled polyclonal antibodyF1031-8-3 antibody antibody (19) Gold colloid- Str Anti-rabbitBio-F1031-8-3 labeled polyclonal antibody S68 antibody antibody (20)F(ab′)₂ of gold F1103-13-3 Anti-rabbit colloid-labeled S68 antibodypolyclonal antibody antibody

By the way, F(ab′)₂ of S68 antibody labeled with gold colloid of (20)<1>is prepared as follows. The preparation of F(ab′)₂ from S68 antibody isperformed as follows using Immobilized Pepsin (PIERCE). That is, S68antibody is dissolved in a 20 mM acetate buffer (pH 4.5) to be preparedto 5 mg/ML. 0.25 mL of Immobilized Pepsin is prepared by suspensionaccording to the protocol of PIERCE and mixed with 1 mL of the aboveantibody. Next, the mixture is stirred for 4 hours in an incubator at37° C., and then the reaction is terminated by the addition of 1.5 mL of10 mM Tris-HCl (pH 7.5). The reaction solution is centrifuged (1000×g)to separate a gel and a supernatant. Then, the separated supernatant isadded to 1 mL of prosep-A (Millipore) to allow the binding of peptidesincluding Fc portion such as Fc fragment and undigested. Likewise, themixture is centrifuged to collect the supernatant, and then thesupernatant is dialyzed against PBS (pH6.4). The absorbance of F(ab)′₂at 280 nm is measured and then the concentration of F(ab)′₂ iscalculated from the absorption constant (0.533 mg/mL/cm⁻¹). Theresulting F(ab′)₂ is labeled with gold colloid as in the case of Example4, resulting in F(ab′)₂ of the gold colloid-labeled S68 antibody.

8-(21) Configuration Example of Flow-Through System

-   <1> Dye-labeled antibody: RED VIOLET dye-labeled S68 antibody-   <2> Conjugate pad: Filter paper (No. 63, manufactured by Advantec    Toyo Co., Ltd.) impregnated with the above (1)<-   <3> Antibody-immobilized membrane: Nitrocellulose membrane (Advantec    Toyo) on which S68 antibody is immobilized-   <4> Absorbing pad: Filter paper (No. 28, Advantec Tokyo) laminated    with polypropylene-   <5> Housing case: Case described in JP 06-273419 A (manufactured by    Mochida Pharmaceutical); <2> to <4> are assembled in <5> such that a    liquid dropped in <2> is allowed to flow through <2>, <3>, and <4>    in this order.

Example 9 Detection of Human Low-Molecular-Weight CD14

(1) Gel Filtration Chromatography <1>

For analyzing the substance in serum of a patient suffering from sepsisdetected by the assay kit described in Example 8-(1), the serum of thepatient suffering from sepsis was fractionated through a gel filtrationchromatography column Superdex 200PC 3.2/30 (Amersham Bioscience) withSMART SYSTEM (Amersham Bioscience) using D-PBS as a elution buffer.Then, each fraction was assayed using the assay kit described in Example8-(1) and the commercially available CD14-EIA kit (IBL-Hamburg). Themolecular weight thereof was calculated by calibrating the column usingaldolase (158 kDa), BSA (67 kDa), ovalbumin (43 kDa), and chymotrypsin(25 kDa) from the LMW calibration kit and HMW calibration kit (AmershamBioscience).

As a result, as shown in FIG. 6, the commercially available CD14-EIA kitdetected soluble CD14 having a molecular weight of about 57 kDa, whichwas defined as high-molecular-weight soluble CD14 of 49 to 55 kDaconventionally reported. On the other hand, in the kit described inExample 8-(1), a peak derived from human low-molecular-weight CD14detected in a patient suffering from sepsis was detected around amolecular weight of 35 to 45 kDa but no peak was detected around 57 kDa.Thus, the result confirmed that the kit described in Example 8-(1)specifically detects only a soluble protein present in blood.

(2) Gel Filtration Chromatography <2>

As in the case of (2)-<1>, 50 μl of serum from a patient suffering fromsepsis was fractionated through a gel filtration chromatography columnSuperdex 75 10/300 GL (Amersham Bioscience) using 200 mM ammoniumacetate (pH 6.8) as a elution buffer and was subjected to the assayusing each kit. The molecular weight thereof was calculated bycalibrating the column using BSA (67 kDa), ovalbumin (43 kDa),chymotrypsinogen (25 kDa), and ribonuclease A (13.7 kDa) from the LMWcalibration kit and HMW calibration kit (Amersham Bioscience).

The results are shown in FIG. 7. In the kit described in Example 8-(1),a peak derived from human low-molecular-weight CD14 was detected arounda molecular weight of 25 to 35 kDa.

(3) F1025-3-1 Antibody Affinity Column Chromatography

When a peaked fraction (e.g., fraction 12) derived from humanlow-molecular-weight CD14 obtained in (2)-<2> is applied to P1025-3-1antibody affinity column chromatography, a peak derived from humanlow-molecular-weight CD14 is eluted in an affinity column non-absorbingfraction. By the way, the adjustment and operation of the F1025-3-1antibody affinity column can be performed on the basis of the methoddescribed in Example 10 of WO 01/22085.

These results show that the human low-molecular-weight CD14 is a solubleprotein in blood that specifically binds to antibodies against aspecific peptide described in SEQ ID NO: 2 having a sequence detectedonly in human CD14 and also binds to an anti-CD14 antibody recognizingan amino acid sequence at positions 17 to 26 from the N-terminal ofhuman CD14. The gel filtration determines the molecular weight thereofto be 25 to 45 kD. Thus, it can be defined that the humanlow-molecular-weight CD14 is smaller in molecular weight than highmolecular-weight CD14 (the conventional native CD14). In addition, thelow-molecular-weight CD14 does not bind to F1025-3-1 antibody thatspecifically binds to high-molecular-weight CD14.

Example 10 Assay of Low-Molecular-Weight CD14 in Sera of PatientsSuffering from Various Kinds of Diseases

10 examples, from which isolates were identified, were used (Table 9) asthe sera of patients suffering from sepsis. In addition, the assay wasconducted using the assay kit described in Example 8-(1) on 52 examplesof normal individuals (male 31 examples and female 21 examples), andpatients suffering from various kinds of diseases (20 diseases, 60examples).

TABLE 9 Number Sex Age Bacteria 1 Male 41 Coagulase-negative bacteria 2Female 44 Coagulase-negative bacteria 3 Female 61 Faecium bacteria 4Male 52 Serratia bacteria 5 Male 37 Escherichia coli 6 Female 67Escherichia coli 7 Male 70 Staphylococcus aureus 8 Male 51 Pantoeaagglomerans 9 Female 81 Escherichia coli 10 Male 77 Escherichia coli

The level of low-molecular-weight CD14 in serum of a normal individualwas in the range of 0.008 to 0.100 μg/mL and the average thereof was0.04 μg/mL. In the case of a patient suffering from sepsis, the level oflow-molecular-weight CD14 was in the range of 0.190 to 7.260 μg/mL andthe average thereof was 2.0 μg/mL. The level of low-molecular-weightCD14 of the patient suffering from sepsis was higher than those of thenormal individuals and patients suffering from other various kinds ofdiseases. Among patients suffering from other various kinds of diseases,there was no patient showing a high level, compared with that of thenormal individual.

Example 11 Comparison with Commercially Available ELISA Kit for CD14Soluble in Blood

11-(1) Assay of Soluble CD14 in Blood of Patients Suffering from VariousKinds of Diseases

Specimens of Example 10 were assayed using the commercially availableCD14-ELISA kit (IBL-Hamburg). The level of soluble CD14 in blood(estimated as a total of low-molecular-weight CD14 andhigh-molecular-weight CD14) of a normal individual was in the range of5.6 to 11.2 μg/mL but an example of a high level in the case of apatient suffering from sepsis was observed. However, many cases thatshowed high levels of soluble CD14 were found in sera of patientssuffering from various kinds of disease, so that there was no differencewith the patients suffering from sepsis.

11-(2) Comparison with Kit Using S68 Antibody

The comparison with and investigation of the measured levels oflow-molecular-weight CD14 determined in Example 11 were performed. Asshown in Table 10, the commercially available CD14-EIA kit showed analmost 1.7-fold difference at maximum among the normal, variousdiseases, and sepsis, while the assay kit of Example 9-(1) showed a50-fold difference between the normal individuals and the sepsis inspite of no difference between the normal individuals and variousdiseases. Therefore, the result was cleared that the measured level ofthe assay kit of Example 9-(1) specifically increases in sepsis,

TABLE 10 CD14 level in blood (μg/mL) Various kinds of Ratio Normaldiseases Sepsis Sepsis/Normal Assay kit of 0.04 0.06 2.0 50.0 Example9-(1) Commercially 7.6 9.0 13.2 1.7 available CD14-EIA

The average level+3 S.D of the tested normal individuals was provided asa cut-off level (low-molecular-weight CD14-EIA: 0.134 μg/mL,commercially available CD14-EIA: 11.14 μg/mL) and then the analyses weredivided into positive samples (sepsis) and negative samples(normal+various diseases). The results were shown in Table 11. Accordingto the results, a rate of identical between both kits ((the number ofidentical for EIA positive+the number of identical for EIAnegative)/total×100), sensitivity (the number of identical for EIApositive/positive examples×100), and specificity (the number ofidentical for EIA positive/negative examples×100) were calculated. As aresult, as shown in Table 12, in the case of low-molecular-weightCD14-EIA, the identical rate was 94.3%, the sensitivity was 100.0%, andthe specificity was 93.8%. Thus, it was found that thelow-molecular-weight CD14-EIA could be useful in differential diagnosison sepsis by defining the cut-off level. On the other hand, in the caseof the commercially available CD14-EIA, there was no sensitivity andspecificity which were specific to allow diagnosis of sepsis.

TABLE 11 Negative sample Positive Various Classification sample kinds ofDisease Sepsis Normal diseases Total Assay kit of 10 51 54 115 Example9-(1) Commercially 6 51 45 102 available CD14-EIA Total 10 52 60 122

TABLE 12 Assay kit of Commercially Example 9 available CD14-EIA Rate ofidentity (%) 94.3% 83.6% Sensitivity (%) 100.0% 60.0% Specificity (%)93.8% 85.7%

INDUSTRIAL APPLICABILITY

According to the present invention, there is provided the antibodyprepared using a peptide as an antigen, the peptide having 8 to 30 aminoacid residues selected from an amino acid sequence at positions 1 to 68of human high-molecular-weight CD14, and also provided the antibody thatbinds to a peptide having amino acid residues described in SEQ ID NOS: 2to 4.

Those antibodies can be used in an assay kit for humanlow-molecular-weight CD14 and the kit is able to quantitatively orqualitatively determine human low-molecular-weight CD14 with highsensitivity in a simple manner, so that the kit is useful for thediagnosis of a patient suffering from sepsis. In the present invention,the assay kit for human low-molecular-weight CD14 containing the aboveantibody and the assay method are provided. Furthermore, the noveldiagnostic method for sepsis in which human low-molecular-weight CD14 isdirectly assayed is provided. Furthermore, the peptide useful in thepreparation of the above antibody and the method of preparing the aboveantibody are provided.

1. A method of detecting human low-molecular-weight CD14 in a specimen,which comprises: contacting the specimen with: (a) an antibody thatbinds to a peptide consisting of the amino acid sequence of SEQ ID No:2;and (b) an antibody that binds to a peptide consisting of the amino acidsequence from position 17 to position 26 of SEQ ID NO:5; wherein saidhuman low molecular weight CD 14: (1) is not bound by F1025-3-1(Accession No. FERM BP-7296) antibody, (2) has a peak elution at amolecular weight range of 25 to 45 kDa as determined by gel filtrationchromatography, and (3) is obtainable from human blood; and detectingbinding of antibodies (a) and (b) to said human low-molecular-weightCD14, whereby said method can detect low-molecular-weight CD14 withoutdetecting high-molecular-weight CD14.
 2. A method of detecting humanlow-molecular-weight CD14 without detecting human high-molecular weightCD14 which comprises: binding said human low-molecular-weight CD14 with(a) an antibody that binds to a peptide consisting of the amino acidsequence of SEQ ID NO:2; and (b) an antibody that competes with anantibody which binds to a peptide consisting of the amino acid sequencefrom position 17 to position 26 of SEQ ID NO:5; wherein said humanlow-molecular weight CD14: (1) is not bound by F1025-3-1 (Accession No.FERM BP-7296) antibody, (2) has a peak of elution at a molecular weightrange of 25 to 45 kDa on a gel filtration chromatography, and (3) isobtainable from human blood; and detecting binding of antibodies (a) and(b) to said human low-molecular-weight CD14, whereby said method candetect low-molecular-weight CD14 without detecting high-molecular-weightCD
 14. 3. A method for diagnosing sepsis in a patient comprising thesteps of: detecting human low-molecular weight CD14 in patient blood bycontacting patient blood with: (a) an antibody that binds to a peptideconsisting of the amino acid sequence of SEQ ID NO:2; and (b) anantibody that binds to a peptide consisting of the amino acid sequencefrom position 17 to position 26 of SEQ ID NO:5; wherein said humanlow-molecular-weight CD 14: (1) is not bound by F1025-3-1 (Accession No.FERM BP-7296) antibody; (2) has a peak of elution at a molecular weightrange of 25 to 45 kDa as determined by gel filtration chromatography,and (3) is obtainable from human blood; measuring in said patient bloodthe amount of low-molecular-weight CD14 bound to both of the abovedescribed antibody (a) and the above described antibody (b), therebydetermining the amount of human low-molecular-weight CD14 in saidpatient blood; comparing the measured amount of low-molecular-weight CD14 in said patient blood to a standard amount of low-molecular-weightCD14 in a normal individual; and evaluating whether the measured amountof human low-molecular weight CD14 observed in said patient blood ishigher than the standard amount of human low-molecular weight CD14observed in a normal individual.
 4. A method for diagnosing sepsis in apatient comprising the steps of: detecting human low-molecular-weightCD14 in patient blood by contacting patient blood with a sandwichimmunoassay kit, wherein said kit comprises: (a) an antibody that bindsto a peptide consisting of the amino acid sequence of SEQ ID NO:2; and(b) an antibody that competes with an antibody which binds to a peptideconsisting of the amino acid sequence from position 17 to position 26 ofSEQ ID NO:5; wherein said human low-molecular-weight CD14: (1) is notbound by F1025-3-1 (Accession No. FERM BP-7296) antibody, (2) has a peakof elution at a molecular weight range of 25 to 45 kDa as determined bygel filtration chromatography, and (3) is obtainable from human blood;measuring in said patient blood the amount of low-molecular-weight CD14bound to both of the above described antibody (a) and the aboveidentified (b), thereby determining the amount of humanlow-molecular-weight CD14 in said patient blood; comparing the measuredamount of low-molecular-weight CD 14 observed in said patient blood to astandard amount of low-molecular-weight CD14 present in a normalindividual; and evaluating whether the measured amount oflow-molecular-weight CD14 observed in said patient blood is higher thanthe standard amount of low-molecular-weight CD14 observed in a normalindividual.
 5. The method for diagnosing sepsis according to claim 3,wherein in said comparing step, the average +2SD of normal individualsis used as a cut-off level.
 6. The method according to claim 1, whereinsaid detecting binding of antibodies (a) and (b) to said humanlow-molecular-weight CD14 is by sandwich immunoassay.
 7. The methodaccording to claim 2, wherein said detecting binding of antibodies (a)and (b) to said human low-molecular-weight CD14 is by sandwichimmunoassay.
 8. The method according to claim 3, wherein said detectingbinding of antibodies (a) and (b) to said human low-molecular-weightCD14 is by sandwich immunoassay.